Rice resistant to HPPD and accase inhibiting herbicides

ABSTRACT

Rice is described that is tolerant/resistant to a plurality of herbicides, for example, ACCase and HPPD inhibitors. Use of the rice for weed control and methods of producing tolerant/resistant rice are also described.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application claims the benefit of priority under 35 U.S.C. §119(e)to U.S. Provisional Patent Application No. 61/869,608, filed Aug. 23,2013, and is a Continuation-in-Part of copending U.S. patent applicationSer. No. 13/975,034, filed Aug. 23, 2013, which claims the benefit ofpriority under 35 U.S.C. §119(e) to U.S. Provisional Patent ApplicationNo. 61/692,861 filed Aug. 24, 2012; this application is also aContinuation-in-Part of copending U.S. patent application Ser. No.13/554,675, filed Jul. 20, 2012, which claims the benefit of priorityunder 35 U.S.C. §119(e) to U.S. Provisional Patent Application Nos.61/510,585, filed Jul. 22, 2011, and 61/541,832, filed Sep. 30, 2011.The disclosures set forth in the referenced applications areincorporated herein by reference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 13, 2014, isnamed 706191_SEQ_US.txt and is 79 KB in size.

BACKGROUND

Mutant rice is disclosed that is (1) resistant/tolerant to both HPPD andACCase inhibiting herbicides; or (2) resistant/tolerant only to4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibiting herbicides.Methods of weed control are disclosed using rice with these herbicideresistant/tolerant crops in fields. Methods to produce herbicideresistant/tolerant rice are also disclosed.

Value of Rice Crops

Rice is an ancient agricultural crop and today is one of the principalfood crops of the world. There are two cultivated species of rice: Oryzasativa L., the Asian rice, and Oryza glaberrima Steud., the Africanrice. The Asian species constitutes virtually all of the worldscultivated rice and is the species grown in the United States. Threemajor rice producing regions exist in the United States: the MississippiDelta (Arkansas, Mississippi, northeast Louisiana, southeast Missouri),the Gulf Coast (southwest Louisiana, southeast Texas), and the CentralValley of California. Other countries, in particular in South Americaand the East, are major rice producers.

Rice is one of the few crops that can be grown in a shallow flood as ithas a unique structure allowing gas exchange through the stems betweenthe roots and the atmosphere. Growth in a shallow flood results in thebest yields and is the reason that rice is usually gown in heavy claysoils, or soils with an impermeable hard pan layer just below the soilsurface. These soil types are usually either not suitable for othercrops or at best, the crops yield poorly.

The constant improvement of rice is imperative to provide necessarynutrition for a growing world population. A large portion of the worldpopulation consumes rice as their primary source of nutrition and cropsmust thrive in various environmental conditions including competing withweeds and attacks by unfavorable agents. Rice improvement is carried outthrough conventional breeding practices and also by recombinant genetictechniques. Though appearing straightforward to those outside thisdiscipline, crop improvement requires keen scientific and artistic skilland results are generally unpredictable.

Although specific breeding objectives vary somewhat in the differentrice producing regions of the world, increasing yield is a primaryobjective in all programs.

Plant breeding begins with the analysis and definition of strengths andweaknesses of cultivars in existence, followed by the establishment ofprogram goals, to improve areas of weakness to produce new cultivars.Specific breeding objectives include combining in a single cultivar animproved combination of desirable traits from the parental sources.Desirable traits may be introduced due to spontaneous or inducedmutations. Desirable traits include higher yield, resistance toenvironmental stress, diseases and insects, better stems and roots,tolerance to low temperatures, better agronomic characteristics,nutritional value and grain quality.

For example, the breeder initially selects and crosses two or moreparental lines, followed by selection for desired traits among the manynew genetic combinations. The breeder can theoretically generatebillions of new and different genetic combinations via crossing.Breeding by using crossing and selfing, does not imply direct control atthe cellular level. However, that type of control may be achieved inpart using recombinant genetic techniques.

Pedigree breeding is used commonly for the improvement ofself-pollinating crops such as rice. For example, two parents whichpossess favorable, complementary traits are crossed to produce an F₁generation. One or both parents may themselves represent an F₁ from aprevious cross. Subsequently a segregating population is produced, bygrowing the seeds resulting from selfing one or several F₁s if the twoparents are pure lines, or by directly growing the seed resulting fromthe initial cross if at least one of the parents is an F₁. Selection ofthe best individual genomes may begin in the first segregatingpopulation or F₂; then, beginning in the F₃, the best individuals in thebest families are selected. “Best” is defined according to the goals ofa particular breeding program e.g., to increase yield, resist diseases.Overall a multifactorial approach is used to define “best” because ofgenetic interactions. A desirable gene in one genetic background maydiffer in a different background. In addition, introduction of the genemay disrupt other favorable genetic characteristics. Replicated testingof families can begin in the F₄ generation to improve the effectivenessof selection for traits with low heritability. At an advanced stage ofinbreeding (i.e., F₆ and F₇), the best lines or mixtures ofphenotypically similar lines are tested for potential release as newparental lines.

Backcross breeding has been used to transfer genes for a highlyheritable trait into a desirable homozygous cultivar or inbred linewhich is the recurrent parent. The source of the trait to be transferredis called the donor parent. The resulting plant is expected to have theattributes of the recurrent parent (e.g., cultivar) and the desirabletrait transferred from the donor parent. After the initial cross,individuals possessing the phenotype of the donor parent are selectedand repeatedly crossed (backcrossed) to the recurrent parent. Theprocess is used to recover all of the beneficial characteristics of therecurrent parent with the addition of the new trait provided by thedonor parent.

Promising advanced breeding lines are thoroughly tested and compared toappropriate standards in environments representative of the commercialtarget area(s) for at least three or more years. The best lines arecandidates for new commercial varieties or parents of hybrids; thosestill deficient in a few traits may be used as parents to produce newpopulations for further selection.

These processes, which lead to the final step of marketing anddistribution, usually take from 8 to 12 years from the time the firstcross is made and may rely on the development of improved breeding linesas precursors. Therefore, development of new cultivars is not only atime-consuming process, but requires precise forward planning, efficientuse of resources, and a minimum of changes in direction. The resultsinclude novel genetic combinations not found in nature.

Some improvement of rice through breeding may be restricted to thenatural genetic variation in rice and hybridizing species, such as wildrice. The introduction of new variation in a breeding program is usuallythrough the crossing program as described, such as pedigree or backcrossbreeding. However, occasionally natural mutations are found that resultin the introduction of new traits such as disease resistance or heightchanges. Breeders have also developed new traits by inducing mutations(small changes in the DNA sequence) into a rice genome. Some of thesemutations or combination of genes are not found in nature. Commonly, EMSor sodium azide plus MNU are used as mutagenic agents. These chemicalsrandomly induce single base changes in DNA, usually of G and C changedto A and T. Overall effects are unpredictable. Most of these changeshave no effect on the crop as they fall either outside the gene codingregions or don't change the amino acid sequence of the gene product.However, some produce new traits or incorporate new DNA changes intoprevious lines.

The breeder has no direct control of mutation sites in the DNA sequence.The identification of useful changes is due to the random possibilitythat an effective mutation will be induced and that the breeder willrecognize the phenotypic effects of the change and will be able toselect rice having that mutation. Seeds are treated with the mutagenicchemical and immediately planted to grow and produce M2 seed. The M2seed will carry numerous new variations; therefore, no two experimentswill produce the same combinations. Among these variations new traitspreviously not existing in rice and unavailable for selection by a plantbreeder may be found and used for rice improvement.

To find new traits the breeder must use efficient and strategicselection strategies as the process is completely random and has anextremely low frequency of useful new combinations. Among thousands ofinduced new genetic variants there may be only one with a desirable newtrait. An optimal selection system will screen through thousands of newvariants and allow detection of a few or even a single plant that mightcarry a new trait. After identifying or finding a possible new trait thebreeder must develop a new cultivar by pedigree or backcross breedingand extensive testing to verify the new trait and cultivar exhibitsstable and heritable value to rice producers.

Using recombinant genetic techniques, nucleic acid molecules withmutations that encode improved characteristics in rice, may beintroduced into rice with commercially suitable genomes. After amutation is identified by whatever course, it may be transferred intorice by recombinant techniques.

Applications of Herbicide Resistance Patents in Rice

Weeds and other competitors for resources in crop fields compete forresources and greatly reduce the yield and quality of the crop. Weedshave been controlled in crops through the application of selectiveherbicides that kill the weeds, but do not harm the crop. Usuallyselectivity of the herbicides is based on biochemical variations ordifferences between the crop and the weeds. Some herbicides arenon-selective, meaning they kill all or almost all plants. Non-selectiveor broad spectrum herbicides can be used in crops if new genes areinserted that express specific proteins that convey tolerance orresistance to the herbicide. Resistance to herbicides has also beenachieved in crops through genetic mutations that alter proteins andbiochemical processes. These mutations may arise in nature, but mostlythey have been induced in crops or in vitro in tissue cultures or byinducing mutations in vivo. Unfortunately in some instances, especiallywith repeated use of a particular herbicide, weeds have developedresistance through the unintended selection of natural mutations thatprovide resistance. When weeds become resistant to a particularherbicide, that herbicide is no longer useful for weed control. Thedevelopment of resistance in weeds is best delayed through alternatingthe use of different modes of action to control weeds, interruptingdevelopment of resistant weeds.

Rice production is plagued by broad leaf plants and a particularly hardto control weed called red rice. One difficulty arises because red riceis so genetically similar to cultivated rice (they occasionally crosspollinate) that there are no selective herbicides available that targetred rice, yet do not harm the cultivated rice. Control is currentlyprovided in commercial rice production through the development ofmutations found in rice that render rice resistant to broad spectrumherbicides e.g. imidazolinone and sulfonylurea herbicides. Riceresistant to herbicides that inhibit other deleterious plants, such asbroad leaf plants, are needed.

Finding new mutations in rice that makes it resistant to a variety ofherbicides, and to combinations of herbicides with alternative modes ofaction, would greatly benefit rice production. Obtaining andincorporating genes for herbicide resistance into rice genomes withadditional favorable characteristics and alternative resistances ischallenging, unpredictable, time consuming and expensive, but necessaryto meet the world's increasing food needs.

SUMMARY

Described and disclosed herein are novel and distinctive rice lines withunique resistances to herbicides in particular HPPD and ACCaseinhibiting herbicides and combinations thereof. For example, a mutantrice line designated ML0831266-03093 is disclosed that isresistant/tolerant to HPPD inhibiting herbicides (ATCC depositPTA-13620). The HPPD inhibiting herbicides include mesotrione,benzobicyclon, and combinations thereof. An embodiment of a mutant riceline designated ML0831265-01493 (ATCC deposit PTA-12933, mutationG2096S) is resistant/tolerant to ACCase inhibitors.

Embodiments of rice resistant to both HPPD and ACCase inhibitors,include rice designated PL121448M2-80048 (ATCC deposit PTA-121362) andPL 1214418M2-73009 (ATCC deposit PTA-121398).

A method to control weeds in a rice field, wherein the rice in the fieldincludes plants resistant to a plurality of herbicides, includes:

-   -   a. using herbicide resistant/tolerant rice in the field; and    -   b. contacting the rice field with a plurality of herbicides, for        example, one of which is an HPPD inhibiting herbicide, another        an ACCase inhibitor.

Rice lines either singly or multiply resistant/tolerant extend theuseful life of several herbicides due to being able to rotate the kindsof herbicides applied in grower's fields thus slowing the development ofweed resistance. Several methods are possible to deploy theseresistances into hybrids or varieties for weed control, as well asoptions for hybrid seed production. The rice lines described hereinrepresent new methods for weed control in rice and can be deployed inany of many possible strategies to control weeds and provide forlong-term use of these and other weed control methods. In particular,mutant rice tolerant to HPPD inhibiting herbicides and to both HPPD andACCase inhibitors are disclosed.

Rice production for good yields requires specific weed controlpractices. Some herbicides are applied at the time of planting andothers are applied before a permanent flood is applied, few weeds cangrow in a full flood.

Through developing sources of resistance to multiple herbicides, moreoptions are available for weed control in rice. The rice lines claimedprovide the ability to use herbicides with a new mode of action for weedcontrol. The ability to use an HPPD inhibiting herbicide in combinationwith an ACCase inhibitor, represents a mode of action not previouslyreported in rice. The use of these rice lines including combining lineswith resistance to herbicide with other modes of action provides newoptions for weed control in grower's fields thus slowing the developmentof weed resistance. Several methods are possible to deploy thisresistance in hybrids for weed control as well as options for hybridseed production.

Cells derived from herbicide resistant seeds, plants grown from suchseeds and cells derived from such plants, progeny of plants grown fromsuch seed and cells derived from such progeny are within the scope ofthis disclosure. The growth of plants produced from deposited seeds, andprogeny of such plants will typically be resistant/tolerant to HPPDinhibiting and ACCase inhibiting herbicides at levels of herbicide thatwould normally inhibit the growth of a corresponding wild-type plant.

A method for controlling growth of weeds in the vicinity of herbicideresistant/tolerant rice plants is also within the scope of thedisclosure. One example of such methods is applying one or moreherbicides to the fields of rice plants at levels of herbicide thatwould normally inhibit the growth of a rice plant. For example, at leastone herbicide inhibits HPPD activity. A plurality includes, for example,HPPD and ACCase inhibitors. Surprisingly, some mixtures of herbicideincreased the activity of all components. Such methods may be practicedwith any herbicide that inhibits HPPD and/or ACCase activity and anyresistant rice mutation, e.g., the embodiments disclosed herein.

Unexpectedly, using a mixture of HPPD and ACCase inhibiting herbicides,provided better results than when each herbicide was applied separately.

A method for growing herbicide resistant/tolerant rice plants includes(a) planting resistant rice seeds; (b) allowing the rice seeds tosprout; (c) applying one or more herbicides to the rice sprouts atlevels of herbicide that would normally inhibit the growth of a riceplant. For example, at least one of the herbicides inhibits HPPD, otherherbicides include ACCase inhibitors.

Methods of producing herbicide-tolerant rice plants may also use atransgenes or plurality of transgenes. One embodiment of such a methodis transforming a cell of a rice plant with transgenes, wherein thetransgenes encode an HPPD and an ACCase enzyme that confers tolerance inresulting rice plants to one or more herbicides. Any suitable cell maybe used in the practice of these methods, for example, the cell may bein the form of a callus.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a graphical representation of a trait tolerance trial; thetrial was planted, mesotrione was applied at the indicated rates about 1month later (3-4 leaf stage), and a second application was applied onthe two indicated treatments about 1 week later; the rice was evaluatedfor the percent of injury or damage as compared to unsprayed ricetwenty-one days after the first herbicide application; lineML0831266-03093 is the HPPD resistant line and R0146 and P1003 arecontrol lines with no induced mutation; the line PL1214418M2-73009 wasselected out of a cross between the HPPD tolerant line ML0831266-03093(parent is P1003) and the ACCase resistant line ML0831265-01493 (parentis R0146). The newly developed line PL1214418M2-73009 shows equivalenttolerance as the original HPPD tolerant line ML0831266-03093.

FIG. 2 is a graphical representation of a trait tolerance trial; thetrial rice were planted, quizalofop was applied at the indicated ratesabout 5 weeks later (initiating tillering); the rice was evaluated forthe percent of injury or damage as compared to unsprayed rice four weeksafter herbicide application; line ML0831265-01493 is the ACCaseresistant line and R0146 and P1003 are control lines; the linePL1214418M2-73009 was selected out of a cross between the HPPD tolerantline ML0831266-03093 (parent is P1003) and the ACCase tolerant lineML0831265-01493 (parent is R0146). The newly developed linePL1214418M2-73009 which has combined resistance shows equivalent orbetter as the original ACCase resistant line ML0831265-01493.

FIG. 3 (A, B) are photographs of line ML0831266-03093 that in additionto resistance to HPPD herbicides also has enhanced resistance to ACCaseherbicides. Enhanced resistance is demonstrated by plants from the HPPDresistant line ML0831266-03093 (A) surviving an application ofquizalofop (77 gm ai/ha) whereas the wild-type parent line P1003 (B)does not survive. Photographs taken four weeks after herbicideapplication.

FIG. 4 is a graphical representation of results of a trait tolerancetrial wherein mesotrione was applied at the indicated rates about 34days after planting. (initiating tillering) the rice was evaluated forthe percent of injury or damage as compared to unsprayed rice four weeksafter herbicide application; the HPPD tolerant line ML0831266-03093 andthe line with combined HPPD and ACCase tolerance PL1214418M2-73009 showa similar response indicating the full HPPD tolerance was recovered inthe new line; the line PL1214418M2-73001 was selected to only carry theHPPD tolerant mutation from the HPPD line ML0831266-03093 and the linePL1214418M2-73013 was selected to only carry the HPPD non-inducedtolerance from the HPPD line ML0831266-03093; P1003 is the non-mutantparent line for the HPPD tolerant line ML0831266-03093; note that over420 gmai/ha, even the lines with genetic resistance may be injured.

FIG. 5 illustrates grass weed control by the ACCase inhibitorquizalofop; control is shown by the prevalent grass weed (dead plants)being killed by quizalofot (77 gm ai/ha) while the resistant lineML0831265-01493 (live plants) were not injured.

FIG. 6 (A, B) are photographs of results of trait tolerance trials; weedcontrol by mesotrione herbicide applied pre-planting was evaluated; theplots were planted with a hybrid of the HPPD tolerant lineML0831266-03093; just before planting, the plot on the right (B)received an application of mesotrione at 210 gmai/ha; the plot on theleft (A) had no herbicide applied either pre-plant or post-emergence;pictures were taken four weeks after planting showing differences inweed appearance.

FIG. 7 (A, B) are photographs of rice growth versus stunted growth in atrait tolerance trial; both plots have rice line P1003 (carries somenon-induced tolerance to HPPD herbicides); the herbicide treatments wereapplied at the initiation of tillering; the photographs were taken fourweeks after herbicide application; only the ACCase herbicide fluazifopwas applied to the plot on the left (A) (210 gmai/ha); at this ratefluazifop was not as active, no killing of the rice plants was observed;the right plot (B) was sprayed with a tank mixture of the ACCaseherbicide fluazifop (210 gmai/ha) and the HPPD herbicide mesotrione (210gmai/ha); combining herbicides improves activity.

FIG. 8 is a graphical representation of results of a trait tolerancetrial quizalofop was applied at the indicated rates about 30 days afterplanting (3-4 leaf stage), and a second application was applied on thetwo indicated treatments. The rice was evaluated for the percent ofinjury or damage as compared to unsprayed rice twenty-one days after thefirst herbicide application; line ML0831265-01493 is the ACCase tolerantline with the G2096S mutation; line ML0831265-02283 is also tolerant toACCase herbicides however the tolerance is not from a mutation in theACCase coding gene, R0146 is the parent line for both of the ACCasetolerant lines; P1003 is a control line.

FIG. 9 shows results of a trait mapping experiment; an F2 population wasderived from a cross between a cytoplasmic male sterile line A0109 andthe ACCase tolerant line ML0831265-0228; the F2 individuals in thepopulation were genotyped, sprayed with quizalofop (116 gmai/ha)twenty-six days after planting, and evaluated for tolerance toquizalofop nineteen days after herbicide application; using QTL mappingsoftware, a major QTL for tolerance was identified on chromosome one(indicated by the arrow).

FIG. 10 is a scattergram showing results of a mutation mappingexperiment; an F2 population was derived from a cross between the ACCasetolerant line ML0831265-02283 and the parent line R0146; the F2population was genotyped, sprayed with quizalofop (116 gmai/ha), andevaluated for resistance to quizalofop; only mutations including ACCaseresistant mutations will be segregating in the population; the SNP indexis a measure of the proportion of sequencing reads that carry avariation from the non-mutant line R0146; a score of one indicates thatall sequencing reads had the variation; 19 variations (mutations) had anindex of one and grouped together on chromosome one (circled) indicatingthe probable location of the tolerance causing mutations. (SEQ ID NOs:208-226)

FIG. 11 shows graphical results of HPPD trait tolerance trials;mesotrione was applied at the 3-4 leaf stage of rice (ML0831266-03093;P1003 and R0146) and evaluated twenty-one days after application forresponse to the herbicide; the rice was evaluated for the percent ofinjury or damage [see DEFINITIONS] as compared to unsprayed rice; thelast two treatments included a sequential application two weeks afterthe first application.

FIG. 12 shows graphical results of HPPD trait tolerance trial; apre-plant application of mesotrione was applied (ML0831266-03093; P1003and R0146) at 210 gmai/ha followed by post-emergent mesotrioneapplications at the 3-4 leaf stage at the indicated rates; the rice wasevaluated for the percent of injury or damage as compared to unsprayedrice twenty-one days after the post-emergent application.

FIG. 13 graphically represents results of trait mapping experiments; anF2 population was derived from a cross between the HPPD tolerant lineML0831266-03093 and the ACCase tolerant line ML0831265-01493; thepopulation was genotyped, sprayed with mesotrione at 105 gmai/ha, andevaluated for tolerance to mesotrione; plants inheriting either thenon-induced tolerance or the mutation tolerance from parent lineML0831266-03093 were expected to live; using QTL mapping software, amajor QTL for tolerance was identified on chromosome two (indicated bythe bold arrow). (X axis=chromosome number in the genome; Y axis=score,correlation with phenotype (resistance)).

FIG. 14 graphically represents results of trait mapping experiments; anF2 population was derived from a cross between the HPPD tolerant lineML0831266-03093 and the ACCase tolerant line ML0831265-01493; thepopulation was genotyped, sprayed with mesotrione at 105 gmai/ha,evaluated for tolerance to mesotrione, and sprayed again with mesotrioneat 630 gmai/ha, and evaluated for tolerance to mesotrione; only plantsinheriting the mutation for tolerance from parent line ML0831266-03093were expected to live; using QTL mapping software a major QTL fortolerance was identified on chromosome one (indicated by the arrow) (Xaxis—chromosome number in the genome; Y-axis=score, correlation withphenotype).

FIG. 15 shows results of mutation mapping experiments; an F2 populationwas derived from a cross between the HPPD tolerant line ML0831266-03093and the parent line P1003; the population was genotyped, sprayed withmesotrione at 840 gmai/ha and evaluated for tolerance to mesotrione;only mutations including the HPPD tolerant mutation segregate in thepopulation; the SNP index is a measure of the proportion of sequencingreads that carry a variation from the line P1003; a score of oneindicates that all sequencing reads had the variation; ten variations(mutations) had an index of one and grouped together on chromosome one(circled) indicating the probable location of the tolerance causingmutation. (SEQ ID NOS: 227-236)

FIGS. 16 (A, B) are a tabular representation of segregants identified ina F4 population; the F4 population was derived from F2 selectionscarrying both HPPD and ACCase tolerance from a cross between the HPPDtolerant line ML0831266-03093 and the ACCase tolerant lineML0831265-01493; each row represents a different line and each column isa molecular marker within the QTL for the HPPD tolerant mutation onchromosome 1.(B) is a continuation of (A) black lines (boundaries)represent results of the genotype of the HPPD tolerant lineML0831266-03093 (20) and the genotype of the ACCase tolerant lineML0831265-01493 (10); observing the tolerance level of these lines tothe HPPD herbicide mesotrione allows the tolerance mutation to be mappedto a specific gene.

FIG. 17 is a tabular representation of segregants identified in a F4population; the F4 population was derived from F2 selections carryingboth HPPD and ACCase tolerance from a cross between the HPPD tolerantline ML0831266-03093 and the ACCase tolerant line ML0831265-01493; eachrow represents a different line and each column is a molecular markerwithin the QTL for the HPPD non-induced tolerance on chromosome two;black lines (boundaries) represent results of the genotype of the HPPDtolerant line ML0831266-03093 (20) and the unshaded represents thegenotype of the ACCase tolerant line ML0831265-01493 (10); observing thetolerance level of these lines to the HPPD herbicide mesotrione allowsthe resistance/tolerance to be mapped to a specific gene.

FIG. 18 (A, B, C) are photographs of controls used in the mesotrioneherbicide bioassay after spraying one application of mesotrione at 105gai/ha followed by a second application of 630 gai/ha; (A) the mutantline ML0831266-03093 shows little damage from the spray application,while (B) the unmutated type P1003 is severely injured (damaged) and (C)the other type of rice, R0146, used to make ACCase mutant lineML0831265-01493 is completely killed.

FIG. 19 (A, B) illustrates rate response of the mutant lineML0831266-03093, the unmutated original (parental) line P1003, and adifferent type of rice R0146; mesotrione was applied across all plotsimmediately following planting at a rate of 210 gai/ha; the responserates were applied at the 2-3 leaf stage; response was recordedtwenty-one days following the foliar application. (A)=initial resultsand (B)=subsequent results.

FIG. 20 shows a DNA sequence for the carboxyl transferease coding regionin the ACCase coding gene; a single nucleotide change (box) that encodesa mutation from G2096S is identified in the mutant line ML0831265-0149which is designated as 09PM72399. (SEQ ID NO: 202) [NIPPONBARE (SEQ IDNO: 200) is a control; R0146 (SEQ ID NO: 201) is the original linetreated with a mitogen to produce a mutation population.

FIG. 21 shows comparison of protein sequences for the carboxyltransferase region of the ACCase gene; the line with code 09PM72399 (SEQID NO: 204) is the line ML0831265-0149; this line shows a change of asingle amino acid (box) at position 2096, relative to Black-Grass; R0146(SEQ ID NO: 204) is the original line treated with a mutagen to producea mutation population. [NIPPONBARE is SEQ ID NO: 203]

FIG. 22 illustrates a response of different germplasms (genotypes) ofrice with different genetic backgrounds to mesotrione applied at the 2-3leaf stage; wherein different rates of mesotrione are applied.

FIG. 23 is a photograph showing phenotypic differences (resistance) ofan F2 population derived from the cross of ML0831266-03093 withtolerance to mesotrione and ML0831265-01493 with tolerance to ACCaseherbicides; mesotrione was applied with one application of 105 gai/hafollowed by a second application of 630 gai/ha; individuals (circled)are selected as inheriting the mesotrione tolerance, whereas the plantsthat did not inherit the tolerance are dead or severely injured.

FIG. 24 is a flow chart of a type of cross used in some embodimentsdisclosed herein.

DETAILED DESCRIPTION Mutation Population and Establishment

A mutation breeding program was initiated to develop proprietaryherbicide resistant/tolerant lines. A permanent mutant population wascreated by exposing approximately 10,000 seeds (estimated by the averageweight of a kernel) of three rice lines including P1003, R0146, andP1062 to both mutagens sodium azide (AZ) and methyl-nitrosourea (MNU).The treated seeds were planted. Individual plants were harvestedcreating 8,281 potentially mutation lines. The lines have beenmaintained as a permanent mutant population for trait screening.

Development of Tolerance to HPPD and ACCase Inhibiting Herbicides byCombining the Tolerances in Lines ML0831266-03093 and ML0831265-01493

Herbicides that target the HPPD enzyme, primarily control broad leafweeds. However trials in rice show prevalent control of grass weeds inrice, including red weedy rice especially with mesotrione herbicide.

On the other hand, the primary weed target of ACCase herbicides ismonocot plants including rice, grass weeds, and red rice. However, someACCase herbicides have lower activity on rice. This weakness is likelytransferred to red rice as the plants are very closely related.Combining the HPPD tolerance and the ACCase tolerance into a single riceline allows a broad spectrum weed control strategy for rice. The HPPDherbicide controls broad leaf weeds and enhances the effect of ACCaseherbicides for control of monocot weeds including red rice.

Combining the HPPD tolerance with the ACCase tolerance into a singlerice line was initiated with the HPPD tolerance mapping project bycrossing the HPPD tolerant line ML0831266-03093 to the ACCase tolerantline ML0831265-01493. In mapping the F2 population plants were selectedfor HPPD tolerance by applying mesotrione first at a low rate (105gmai/ha) followed by a high rate (630 gmai/ha). In this processmolecular markers were also developed allowing future selection of HPPDtolerance by either markers or herbicide tolerance screening or both.

After identifying plants that were tolerant to the HPPD herbicidemesotrione, they were also tested with the ACCase tolerance functionalmarker for the G2096S mutation in the ACCase donor parent lineML0831265-01493. Information to develop ACCase G2096S markers are inFIGS. 20, 21.

After this process, a set of 25 F2 plants with the ACCase mutation toherbicide resistance, and the HPPD genetic herbicide resistance onchromosome 1 and chromosome 2, in at least the heterozygous condition,were identified. The plants were transplanted to another field forharvesting at maturity. Out of the 25 plants, eight were homozygous forthe ACCase mutation and one plant was homozygous for the ACCasemutation, the HPPD tolerance mutation, and the non-induced tolerancegene. The 25 plants were bagged at flowering and the seed harvested atmaturity from each plant individually.

An early maturing group of plants was harvested as early as possible andthe seeds planted in the greenhouse to help quickly advance to the F4generation. Selections on the F3 plants were made by molecular markersflanking the HPPD tolerance mutation and native tolerance the ACCasefunctional mutation. Homozygous plants for all the selected genomicregions were advanced to the F5 generation. The F5 seed was confirmed tocarry tolerance to ACCase herbicides and the HPPD herbicide mesotrione.Among the F5 lines PL1214418M2-80048 was selected due to a high seedyield and being homozygous for the ACCase tolerance mutation at positionG2096S, the HPPD tolerance mutation, and the HPPD tolerance native gene.Seed from the line PL1214418M2-80048 was deposited at the ATCC and givena deposit number PTA-121362 (see Table 8).

A second line was developed by planting F3 seed in rows. The plants weresprayed with the HPPD herbicide mesotrione and selected for little or noinjury as compared to unsprayed controls. Leaf tissue was also collectedand the plants were tested for inheritance of the ACCase tolerancemutation G2096S, the HPPD mutation tolerance, and the HPPD non-inducedtolerance. Plants homozygous for all three tolerance genes or QTLs wereidentified and harvested. The F4 seed (PL1214418M2-73009) was bulkedtogether from plants carrying all three tolerance genes or QTLs and usedfor testing or as a new donor line for tolerance to both ACCase and HPPDherbicides. Seed of the source PL1214418M2-73009 was deposited at theATCC and given a deposit number PTA-121398 (see Table 8).

Tolerance of New Lines Combining HPPD and ACCase is Equivalent andSelectable in Breeding Populations

The seed source PL1214418M2-73009 was developed from a cross between theHPPD resistant line ML0831266-03093 and the ACCase resistant lineML0831265-01493 and was sufficient to allow testing to verify equivalenttolerance to HPPD and ACCase inhibitors in new lines. Two trials wereconducted to measure recovery of tolerance to both ACCase and HPPDherbicides in the new line PL1214418M2-73009. Recovery of tolerance inthe line combining the two traits will illustrate that the traits areheritable and can be used to produce new varieties and hybrids carryingherbicide resistance. These trials are important as often times it isdifficult to recover complex QTLs for quantitative traits or in somecases a traits response is dependent upon the genetic background. In thefirst trail the lines resistance to mesotrione (HPPD herbicide) wasevaluated by planting line PL1214418M2-73009, the HPPD resistant lineML0831266-03093 and wild-type rice line P1003 and R0146 in plots (5feet×10 feet). Mesotrione was applied at 0.5×, 1×, 2×, and 4× multiplesof the labeled application rate (210 gmai/ha). Two additional treatmentswere included with a 1× and 2× rate followed by a second application 14days afterward with the same rates. Full recovery of the HPPD resistancefrom line ML0831266-03093 was achieved in the line PL1214418M2-73009 asit and the original trait line had the same response to the herbicideapplications (FIG. 1). These results show that the HPPD resistant traitcan be bred and selected to develop commercial products.

Another trial was conducted to confirm recovery of the ACCase inhibitorresistance from the G2096S mutation as in the line ML0831265-01493. Inthis trial the new line PL1214418-73009 with combined HPPD and ACCasetolerance was planted in a row along with other various lines includingthe original donor line ML0831265-01493 (planted in a plot),ML0831266-03093, P1003, R0146 parent line for ACCase tolerance. Thelines were all tested with the ACCase herbicides fluazifop at 0.5×, 1×,2×, and 4× multiples of the label application rate (210 gmai/ha) andquizalofop at 0.5×, 1×, 2×, and 4× multiples of the label applicationrate (77 gmai/ha). In these trials the three new lines that inheritedthe ACCase tolerance all showed equivalent tolerance to the ACCaseherbicides as did the donor line ML0831265-01493 (FIG. 2).

The tolerance to HPPD herbicides is more complex than the ACCasetolerance because it requires two genes that are different from the genetargeted by the herbicide. In spite of this greater complexity, theequivalent tolerance was recovered through selection of both the nativetolerance gene and the mutation tolerance. The ACCase parent lineML0831265-01493 in this cross was highly sensitive to the HPPD herbicidemesotrione and thus was not expected to contribute any towards HPPDtolerance. Resistance/tolerance to HPPD herbicides is mostly likelycaused by these two genes alone as they were the focus of the selectionprocess, and the new line PL1214418M2-73009 shows equivalent resistance.These results show that the resistance for both ACCase and HPPDinhibitors is inherited and can be bred into any rice for commercialdevelopment of both HPPD and ACCase inhibitor resistance in rice.

Identification of Unexpected Increased Tolerance to ACCase Herbicides bythe HPPD Tolerance Mutation

During the process of showing equivalent resistance of the ACCasetolerance in the new line PL1214418M2-73009, the HPPD tolerant lineML0831266-03093 was also evaluated for response to ACCase tolerance byapplication of the ACCase herbicides fluazifop at 0.5×, 1×, 2×, and 4×multiples of the label application rate (210 gmai/ha) and quizalofop at0.5×, 1×, 2×, and 4× multiples of the label application rate (77gmai/ha). In the field trials the resistant HPPD line ML0831266-03093was planted in a row adjacent to the parent line P1003. Duringobservations it became clear that the line ML0831266-03093 had moreresistance to the ACCase inhibiting herbicides than did line P1003 (FIG.3). These results suggest that the HPPD tolerance mutation has activityagainst ACCase inhibiting herbicides in addition to HPPD inhibitingherbicides. By combining the HPPD tolerance with the ACCase tolerancethe newly developed line PL1214418M2-73009, or any other new line andother derived lines because resistance is heritable and can be bred intolines e.g. progeny may carry a higher tolerance to ACCase inhibitingherbicides than lines developed from only the ACCase inhibitor resistantline ML0831265-01493.

Identification of the Tolerance Contribution from the HPPD ToleranceMutation and the Non-Induced Tolerance Gene from P1003

During the breeding process to develop new lines (PL1214418M2-80048 andPL1214418M2-73009) with resistance/tolerance to HPPD and ACCaseherbicides, two other lines were also investigated to determine thecontribution of the HPPD tolerance mutation and the HPPD tolerancenative gene. The line PL1214418M2-73001 carries ACCase tolerance andonly the HPPD tolerance, whereas mutation PL1214418M2-73013 carriesACCase tolerance and only the HPPD native tolerance gene. Theseselections allow the estimation of the tolerance effect of each of thetwo genes required for tolerance to HPPD herbicides. The toleranceeffect of each gene was measured by growing the lines in single rowsincluding the newly developed line PL1214418M2-73009 that carries boththe HPPD tolerance from the mutation and the non-induced tolerance, theHPPD tolerant line ML0831266-0309, and the non-induced parent lineP1003. The field plots were sprayed at the 4 leaf stage with the HPPDherbicide mesotrione at 0.5×, 1×, 2×, and 4× multiples of the labeledapplication rate of 210 gmai/ha.

The plots were evaluated 4 weeks after the herbicide was applied. Theresults showed that the native tolerance gene alone (PL1214418M2-73013)gave tolerance levels similar to the parent line P1003 (FIG. 4). Thisresult would be expected if the native tolerance gene located onchromosome 2 located within the QTL flanking markers is the onlycausative source of tolerance in the non-mutant line P1003. The linePL1214418M2-73001 that carries only the HPPD mutation located onchromosome 1 within the QTL flanking markers, shows intermediatetolerance between the non-mutant line P1003 and the HPPD mutant lineML0831266-0309. This result shows that the HPPD mutation provides notonly enhanced tolerance but also a greater level of tolerance than thenative tolerance gene. In addition it also suggests that the HPPDmutation functions independently of the HPPD native tolerance gene. Thetwo genes also appear to function in an additive manner as only bycombining the two in the new line PL1214418M2-73009 does the tolerancelevel become equivalent to the original mutant line ML0831266-0309.

Controlling Weeds and Red Rice in Rice Crops with ACCase Inhibitors andMesotrione Herbicides (HPPD Inhibitors)

The herbicide activity or ability to control non-mutant rice, such asline R0146 and P1003, is a good predictor of how well the herbicideswill control red rice or wild weedy rice in a rice crop. Red rice andwild weedy rice are very similar to rice, even with the ability to crosswith rice. This similarity is the reason these weeds are so difficult tocontrol in a rice crop. The mutant lines (ML0831265-01493,ML0831265-02283, ML0831266-03093, PL1214418M2-80048, andPL1214418M2-73009) disclosed offer a new weed control strategy for redrice, wild weedy rice, and other weeds common in rice crops. These linesgive rice tolerance to herbicides that will normally kill or cause yieldreducing injury to the rice crop.

While testing the tolerant lines, the parent lines were also tested toserve as controls and as an indication of commercial potential as a redrice/wild weedy rice control strategy. These trials showed that selecttreatments of the herbicides applied alone or in various combinationsand application timings offer a new weed control strategy in rice crops.

Rice is tolerant to certain ACCase inhibitor herbicides, for examplecyhalofop is registered for use in rice. However other ACCase herbicideskill or severely injure rice to varying degrees. After testing, severalof these other herbicides including fluazifop and quizalofop were foundto offer good control of common grass weeds, such as barnyard grass, inrice (FIG. 5). Control of common weeds in rice was also achieved withmesotrione alone, especially when applied pre-plant or at higher rates(2× the labeled rate of 210 gmai/ha) (FIG. 6). The applied rates of bothtypes of herbicides giving the weed control are well within thetolerance level of the respective ACCase and HPPD tolerant linesincluding the combined lines carrying tolerance to both HPPD and ACCaseherbicides.

Development of the HPPD and ACCase tolerance into single lines(PL1214418M2-80048 and PL1214418M2-73009) gives the opportunity for anadditional weed control strategy involving applications of ACCase andHPPD herbicides in a tank mix or individually at different times. Thevery effective pre-plant application of the HPPD herbicide mesotrionecan now be followed with ACCase herbicides applied alone or incombination with HPPD herbicides. This strategy provides full spectrumweed control in a rice crop by broad leaf weed control provided by theHPPD herbicide, and grass weed control by the ACCase herbicide. Inaddition the control of grasses and red rice/weedy rice by ACCaseherbicides is greatly enhanced by the activity provided by the HPPDinhibiting herbicide. This strategy is anticipated as being especiallyeffective for control of red rice when an ACCase inhibiting herbicidesare used that have lower activity on rice (FIG. 7).

This particular weed control system is highly useful in rice crops dueto some weeds, including red rice, developing tolerance to currentlyused herbicides. Use of this weed control strategy allows rotation ofdifferent modes of action herbicides in rice crops. By rotatingdifferent modes of herbicide action the development of resistant weedsis slowed or prevented allowing for longer term use of all availableweed control methods.

Tolerance/Resistance to ACCase Inhibitors

1. Validation of the Mutant Line ML0831265-02283 for Tolerance to ACCaseHerbicides

After screening a large mutant population, the line ML0831265-02283 alsosurvived application of the ACCase herbicide quizalofop. The line wasincreased to obtain sufficient seed for larger trials to evaluate itstolerance to ACCase herbicides. The tolerance to ACCase herbicides inline ML0831265-02283 was validated by planting in the field plots (5feet by 10 feet) of the line, the non-mutant parent line R0146, a secondnon-mutant line P1003, and the ACCase tolerant line ML0831265-01493. TheACCase herbicide quizalofop was applied at the four leaf stage at 0.5×,1×, 2×, and 4× multiples of the labeled rate (77 gmai/ha). Twenty onedays after the herbicide was applied the plots were evaluated forpercent injury caused to the rice based on control plots that had noherbicide application (FIG. 8). The data confirms the tolerance of lineML0831265-02283 and it may even carry more tolerance than lineML0831265-01493 as shown by less injury at the 2× and 4× rates ofquizalofop.

2. Identification of the Causal Mutation for Tolerance to ACCaseHerbicides in Line ML0831265-02283

Often tolerance to ACCase herbicides is derived from a mutation in thecarboxyl transferase region of the ACCase gene, as is the case intolerant line ML0831265-01493 (mutation at G2096S). However aftersequencing the carboxyl transferase region of the ACCase gene in lineML0831265-02283 no mutation was found. This result indicates that thetolerance in line ML0831265-02283 is derived from a non-target siteprocess.

Finding the causal mutation for tolerance in line ML0831265-02283involved linkage mapping and mutation mapping as (“mut mapping”)described for finding the causal mutation and native tolerance for HPPDtolerance in line ML0831266-0309. (Wright et al., 2011)

Linkage mapping to find the chromosomal region or QTL causing thetolerance in line ML0831265-02283 requires a population segregating forthe trait. This population was made by crossing the tolerant line withthe male sterile cytoplasm line A0109. The F1 collected from this crosswas grown and allowed to self-pollinate to make a F2 population. The F2population will segregate for ACCase tolerance. Eight hundred F2 seedswere planted and leaf tissue was collected from the seedlings to allowgenotyping of each plant. When the seedlings where three weeks old thewhole F2 population was sprayed with quizalfop (116 gmai/ha). Theseedlings were evaluated for tolerance nineteen days after the herbicideapplication. Standard QTL mapping software was used to analyze thegenotypes of each F2 individual and the associated tolerance response toidentify molecular markers linked to the herbicide tolerance. After thisanalysis a genomic region (QTL) was identified for the tolerance onchromosome one (FIG. 9). Linked markers flanking the QTL and markersinside the QTL flanking the peak of the QTL were identified as beingsuitable to select the herbicide tolerance derived from lineML0831265-02283 (TABLE 5). Approximately 250 genes are between theflanking markers.

The mutation mapping strategy to find the causal mutation was employedin the same manner as used to find the QTL for HPPD mutation tolerance.A mutation mapping population was created to find the causal tolerancemutation through genomic sequencing by next-generation sequencing. Themutant line ML0831265-02283 was crossed back to the original non-mutantparent R0146. The F1 progeny of the cross were selfed to produce a F2population that is segregating for the tolerance causing mutation. Onlymutations are segregating in this population because the mutations arethe only genomic difference between ML0831265-02283 and R0146.

The F2 population was planted as individuals, and leaf tissue wascollected and DNA extracted from each individual to use for genotypingafter the population was phenotyped. The ACCase herbicide quizalofop wasapplied to the F2 population at the 3-4 leaf stage and a concentrationof 116 gmai/ha. Individuals that survived the herbicide application werescored as tolerant and those that died were scored as susceptible.

The DNA derived from a set of twenty surviving F2 individuals and twentythat were killed was each respectively bulked together and sequencedalong with both the mutant line ML0831265-02283 and the non-mutantparent line R0146. Mapping the causal mutation was based on an indexaccessing the frequency of all mutations in the bulk representing thesurviving individuals. The index was derived from the proportion ofsequencing reads that carried a variation different from the non-mutantparent line R0146. The more sequencing reads with the variation thecloser the index was to one and if all sequencing reads had thevariation the index equaled one.

The analysis of these results showed two groups including 19 mutationsof eleven mutations on chromosome one with an index score of one (FIG.10). This result confirms the QTL linkage mapping results as themutations identified here all are located within the QTL regionidentified on chromosome one by linkage mapping. Molecular markers (SNP)were made for each of the mutations (TABLE 6). These markers are used infine mapping to find the causal mutation and for breeding the ACCasetolerance derived from line ML0831265-02283 into commercial rice lines.

Tolerance/Resistance to HPPD Inhibitors: Herbicide Screening

Mesotrione (Callisto®), is an herbicide that inhibits the plant enzyme4-hydroxyphenylpyruvate dioxygenase (HPPD). Callisto® is a postemergentand preemergent herbicide used to control annual broadleaf weeds in cornand certain other crops. The herbicide only damages some rice at lowerrates but kills other types of rice. All rice appears to be at leastdamaged by higher herbicide rates. Finding resistance to Callisto®herbicide in rice results in a new mode of action for controllingbroadleaf weeds and some grasses, in rice.

Resistance to mesotrione herbicide was found by screening the permanentmutant population. All lines in the permanent mutant population wereplanted into a dry seed bed. Within twelve hours after plantingmesotrione (Callisto®) was applied at a rate of 255.1 gm ai per acre.The field was immediately flushed with water and kept moist throughperiodic flushing. The seedlings grew, many were bleached white and alllines derived from R0146 died whereas plants lived from 21 lines derivedfrom the P1003 and 2 derived from P1062 mutation populations. The HPPDgene was sequenced, and no genetic mutation was identified causing anyamino acid substitutions (SEQ ID NOs: 1, 2, 3).

Validating the Mutant Line ML0831266-03093 for Tolerance to HPPDInhibiting Herbicides

After the initial screening of the “mutation population,” the lines withno damage were selected and tested in additional experiments usingdifferent rates of the herbicide. In particular, a rate responseexperiment was conducted in which two different rates of mesotrione wereapplied pre-emergence, plus an additional foliar application was alsoapplied. This experiment differentiated one mutant line as havingsuperior resistance (less injury) to the mesotrione herbicide ascompared to the control (FIG. 19, Table 1). Progeny of this designatedML0831266-03093 are maintained as a new line carrying resistance tomesotrione herbicide. Seeds are deposited under ATCC PTA-13620. The line(ML0831266-03093) is a source of resistance that is backcrossed intoproprietary rice lines or used directly in breeding to develop newproprietary rice lines. The developed lines are a source of herbicideresistance for use in development of new hybrids that offer analternative mode of action to control weeds in rice. Affording thisopportunity to growers is of great value both in providing high yieldsand in extending the useful life of available weed control technologies.These herbicide resistant traits can be tracked through the simpleapplication of herbicides to growing plants.

The mutant line ML0831266-03093 was found to carry tolerance tomesotrione (a common HPPD inhibiting herbicide) through screening theline with different rates of the herbicide. The tolerance level ofML0831266-03093 was found to be much greater than the originalnon-mutant (native) line P1003.

The original line P1003, carries natural tolerance to mesotrione. Thisnon-induced resistance of the original line sometimes masked theresistance of the mutant line making the enhanced resistance of themutant line ML0831266-03093 not obvious. Finding the value of the mutantline ML0831266-03093 was only achieved with careful testing over twoyears, in different locations, and using different rates and timings ofherbicide application. The high tolerance of the mutant lineML0831266-03093 is now apparent and documented through a rate responsetrial measuring the response of line ML0831266-03093 and non-mutantcontrol lines to different rates of mesotrione (FIG. 11).

The validating trials included testing applications of mesotrioneapplied just before planting (pre-plant applications), after planting atvarious stages of rice growth (post applications), and combinations ofboth pre-plant and post applications. The discovered tolerance to HPPDinhibiting chemicals was apparent for both pre-plant and post-emergentapplications (FIG. 12). The mutant line ML0831266-03093 shows tolerancegreater than natural rice has to HPPD herbicides in all applicationregimes.

Further validation of the trait involved testing the mutant lineML0831266-03093 in the presence of common rice weeds. The mutant linewas completely tolerant to the applied rates of mesotrione whereas theprevalent weed population was well controlled by the herbicide (FIG. 6).This level of grass weed control was completely unexpected as mesotrioneis labeled for controlling broadleaf weeds in monocot crops. This resultindicates that mesotrione and other HPPD inhibiting herbicides incombination with line ML0831266-03093 and derived lines, represent a newweed control system in rice. The high activity of the mesotrione ongrass weeds and certain types of rice indicates that the system could beused to control red rice in a rice crop.

Mesotrione and other HPPD inhibiting herbicides target the HPPD gene. Anincrease in herbicide tolerance could be achieved through a mutation inthe HPPD gene. A mutation within the gene sequence can alter the enzymestructure sufficiently to prevent it from being inhibited by theherbicide, but still allow it to carry-out its normal physiologicalfunction. Assuming this as a plausible tolerance mechanism, the HPPDgene was sequenced by Sanger sequencing in both the mutant lineML0831266-03093 and the original line P1003. Surprisingly, no mutationwas found in the HPPD gene (SEQ ID NO: 1). The herbicide tolerance inline ML0831266-03093 appears to be derived from a non-target siteprocess.

Two different methods were used to find the tolerance causing mutation.The first method involved using a QTL mapping strategy only employing aunique phenotyping process to find both the tolerance causing mutationand the gene causing the natural tolerance. The second method involvedsequencing the entire genome of F2 plants derived from a cross to thenon-mutant parent. [P1003]

1. QTL Mapping to Find the Causal Mutation and the Natural ToleranceCausing Gene

The mutant line ML0831266-03093 contains high tolerance to mesotrioneand possibly other HPPD inhibiting herbicides, due to both a newmutation and a native tolerance gene present in the original non-mutantline P1003. The mesotrione tolerant line ML0831266-03093 was crossed toanother mutant line ML0831265-01493. This second mutant lineML0831265-01493 lacks the native tolerance gene and is highlysusceptible to mesotrione. However line ML0831265-01493 does havetolerance to ACCase herbicides due to a mutation in the ACCase gene thatchanges amino acid 2096 from glycine to serine. This mutation alters theenzyme making it unaffected by certain ACCase herbicides, however itstill retains its normal physiological function. The mutation site forchange the amino acid 2096 most commonly arises in weeds as a change toalanine rather than the only rarely found serine change. (FIGS. 20, 21)A molecular marker was developed based on the sequencing information, totest for inheritance of the mutation G2096S.

The F1 progeny from the cross of line ML0831266-03093 to ML0831265-01493was selfed to produce a large population of F2 individuals. Each F2individual was genotype with a set of 192 SNP markers (Table 3) thatwere polymorphic between the parents, to fully cover the genome withmolecular markers.

Next in a QTL mapping strategy was to spray the herbicide on the F2individuals and observe those that survive. However, this strategy couldintroduce complications due to both the native tolerance gene and thetolerance mutation segregating. A different strategy was used in whichfirst mesotrione was applied at a low rate (105 gmai/ha). At this rateall plants inheriting the tolerance causing mutation, the nativetolerance, or both, survived, whereas plants inheriting thecorresponding genomic regions from the line ML0831265-01493 died as theyare highly sensitive to the herbicide. QTL analysis based on thisphenotyping method identified one QTL located on chromosome 2 (FIG. 13).After phenotyping the F2 plants with a low application rate ofmesotrione a second high rate (630 gmai/ha) was applied, and the plantswere again phenotyped. After analysis, a second QTL was found onchromosome 1 representing the higher tolerance achieved from thetolerance mutation (FIG. 14). With this strategy the two genes wereresolved, one of which is the mutation (chromosome 1) and the other thenative tolerance (chromosome 2).

Based on the QTL positions and linked markers a set of markers wasidentified that flank the mutation and native tolerance QTLs (TABLE 4).These markers define the region containing the HPPD tolerance causinggenes. In addition this set of markers can be used for breeding purposesto develop new lines carrying tolerance to HPPD herbicides. The use ofthese markers allows selection without having to apply the herbicide tobreeding populations.

2. Mutation Mapping to Find the Tolerance Causal Mutation

A mutation mapping population was created to find the tolerance causalmutation through genomic sequencing by next-generation sequencing. Themutant line ML0831266-03093 was crossed back to the original non-mutantparent P1003. The F1 progeny of the cross was selfed to produce a F2population that will be segregating for the tolerance causing mutation.Only mutations will be segregating in this population because themutations are the only genomic difference between ML0831266-03093 andP1003.

The F2 population was planted as individuals and leaf tissue collectedand DNA extracted from each individual to use for genotyping after thepopulation was phenotyped. In this method all of the population willcarry the native tolerance gene rendering the population tolerant to acertain level to mesotrione herbicide. To differentiate the nativetolerance from the tolerance causal mutation mesotrione was applied tothe population with a high rate (840 gmai/ha) so that all individualswithout the tolerance causal mutation died.

The DNA derived from a set of twenty surviving F2 individuals and twentythat were killed was each respectively bulked together and sequencedalong with both the mutant line ML0831266-03093 and the non-mutantparent line P1003. Mapping the causal mutation was based on an indexaccessing the frequency of all mutations in the bulk representing thesurviving individuals. The index was derived from the proportion ofsequencing reads that carried a variation different from the non-mutantparent line P1003. The more sequencing reads with the variation thecloser the index was to one and if all sequencing reads had thevariation the index equaled one.

A single mutation causing the high tolerance to mesotrione waspredicted. Instead the data showed a peak of mutations carrying a scoreof 1 introducing another level of difficulty in finding the causalmutation. The result did confirm that the QTL on chromosome 1 foundthrough linkage mapping is the genomic location of the tolerance casualmutation (FIG. 15). Within the peak of mutations we found sevenmutations with an index of one, none of which are an obvious cause fortolerance to mesotrione herbicide. (TABLE 7) Markers were developed forthe mutations to facilitate finding the casual resistance mutation(s).

A set of lines was identified with recombination points evenlydistributed within the identified QTLs and mutations (FIG. 16, FIG. 17).These lines were recovered in a homozygous condition for eachrecombination allowing phenotyping for herbicide tolerance on multipleindividuals (full plots). Analysis of these lines allowed the tolerancemutation and native gene to be narrowed to a small region of thechromosome.

Through the described strategy the specific genomic regions containingthe tolerance causal mutation and the native tolerance gene are nowknown and useful for developing commercial products. The commercialproducts are useful in rice production as they survive application ofmesotrione herbicide at rates that will control prevalent weedsincluding red rice without harming the rice crop. The specific genomiclocation allows the use of molecular markers on the flanking regions ofeach QTL to select for the HPPD tolerant trait in the development ofcommercial products.

Genetic mapping of the three groups of F2 individuals including the setof individuals sprayed with mesotrione at only 105 gm ai/ha, the setfollowed by a sequential application of 630 gm ai/ha, and the finalgroup sprayed with 420 gm ai/ha shows two genes controlling resistanceto mesotrione. In the population sprayed with 105 gm ai/ha a single QTLfound on chromosome 2 with strong linkage to SNP marker BG-id2004662acted in a mostly dominate manner. This marker and QTL identifies theinherent tolerance in line P1003. The marker is useful for breeding andselection of new mesotrione tolerant lines. The discovery of this QTLfacilitates commercial development of new rice varieties with a newmethod for controlling weeds through the use of mesotrione herbicide.The finding of the linked marker BG-id2004662 is a novel finding andselection strategy for breeding and selecting the tolerance tomesotrione and other herbicides derived from line P1003.

In the two groups of F2 individuals sprayed with the higher rates ofmesotrione (420 and 630 gm ai/ha) a second QTL was found with stronglinkage to SNP marker WG-id1002788. This QTL is the demonstrated geneticposition of the causal mutation for high tolerance to mesotrione. Thecombined tolerance of the QTL developed through mutation breeding onchromosome 1 and the QTL discovered in line P1003 provides a noveltolerance to mesotrione and combined with the linked molecular markersfacilitates quick and efficient breeding of new rice varieties (FIG.24).

Genetic mapping for the genomic location for resistance in lineML0831266-03093 is carried out by common QTL mapping strategies. Theresistant line ML0831266-03093 may be crossed with a highly sensitiveline and the resulting F1 seeds grown and plants selfed to produce a F2mapping population segregating for the resistance trait. Finding traitlinked markers is done by genotyping each F2 plant, spraying the plantswith an appropriate concentration of the herbicide, and associating themolecular genotypes with the phenotype of each individual. This processwill identify a genomic region between markers for the causal mutationfor resistance.

Identifying the mutation causing the herbicide resistance is possiblethrough a variety of processes. The mutant line and the originalnon-mutant line sequences are prepared and compared to identifymutations within the region of the QTL found through common traitmapping methods. Then markers are developed to the sequence differences,they are testing on a phenotyped segregating population such as the oneused for QTL identification or make a new similar population. In anothermethod, next generation sequencing is used to sequence and compare abulk of individuals that are resistant to either a bulk of susceptibleindividuals or to the original non-mutant line. In this method thecausal mutation is found through the resistant bulk having the highestportion of the mutation or sequence difference as compared to thesusceptible bulk or non-mutant original line.

A recent publication for these methods includes Akira Abe, et al.;Nature Biotechnology 30, 174-178 (2012) doi:10.1038/nbt.2095, Publishedonline 22 Jan. 2012.

EXAMPLES Example 1 Production of Hybrid Rice Resistant to One HPPDInhibiting Herbicide

The HPPD inhibiting herbicide resistance provided by ML0831266-03093 isdeployed individually into hybrids through either the male or femaleparent resulting in the hybrid seed being resistant to the herbicide. Ifthe resistance is deployed in only the male parent, then in addition toits use for weed control, the herbicide when applied to hybrid seedkills contaminating female selfed seed. On the other hand if theresistance is deployed only through the female parent, growers mayeliminate contaminating male selfed seed.

Growers may alternate the type of resistance they purchase and apply intheir fields to reduce the chance that weeds develop resistance to theherbicide. The HPPD inhibiting herbicide, though primarily for controlof broad leaf weeds, also allows for some enhanced control of red rice.At higher rates it will kill certain types of rice. If resistance arosein red rice from cross pollination, it could still be controlled with adifferent herbicide class in the next season.

Example 2 Production of Hybrid Rice with High Level of Resistant to HPPDInhibiting Herbicides

The HPPD inhibiting herbicide resistance provided by ML0831266-03093 isdeployed into both the male and female parents of a hybrid. Theresulting hybrid seed may carry resistance to mesotrione and other HPPDinhibiting herbicides. Resistance provided in this manner is strongerand offers better weed control through the possibility of being able toapply higher rates of herbicide.

Example 3 Production of Hybrid Rice Resistant to Multiple Herbicides

The herbicide resistance for at least 2 herbicides is deployed in asingle hybrid through making both the female and male parent resistantto both herbicides. Deployment in this manner results in hybrid seedbeing homozygous for both resistances. By providing resistance inhomozygous condition in the hybrid for both herbicide classes the hybridseed shows maximum level of resistance. In addition by deploying bothresistances together, the grower has the option to select eitherherbicide to apply in a given season, alternatively, both herbicidescould be applied within the same season. The ability to rotateherbicides provides the opportunity to extend the life of the herbicidesthrough delaying the development of weed resistance. This method alsoallows for the use of both herbicide classes for weed control duringhybrid seed production.

Example 4 Production of Hybrid Rice Resistant to Mesotrione and at LeastOne Other Herbicide Class

1. Resistance to mesotrione and at least one other herbicide class isdeployed in a single hybrid by using a male parent that carriesresistance to the mesotrione (or the other herbicide) and a female thatcarries the other resistance. The method allows the grower to make asingle purchase but to be able to choose which herbicide to apply. Asingle class of herbicide may be used in any one season and rotatedbetween seasons, or alternatively both herbicides could be appliedwithin a single season. In addition, deployment by this method, elementscontaminating selfed seed of both parents in the hybrid seed throughapplication of both herbicides, or one type or the other, are eliminatedthrough application of only one herbicide.

2. In another method of deployment the mesotrione resistance and anyother herbicide resistance is deployed through making a hybrid with amale parent that carries both resistances. The grower then has theoption to choose which herbicide class to apply or to apply both withina single season. In addition, through the application of eitherherbicide contaminating selfed female seed would be eliminated.Alternatively both herbicide class resistances are provided in thefemale parent, giving the grower the same options for weed control.

3. Another embodiment is to deploy the mesotrione resistance to bothparents, and another herbicide resistance into only one parent, such asthe male parent. The hybrid seed are then homozygous for the mesotrioneresistance but not the other. A scheme like this is used to make anearly application with the herbicide put into only the male parent,providing weed control and elimination of contaminating female selfs.Later in the season mesotrione may be applied or another HPPD inhibitorherbicide. The useful life of both herbicides is extended throughlimiting or eliminating the development of weed resistance. In anotherapplication this method allows the use of mesotrione or other HPPDinhibitor herbicide to control weeds in seed production fields, allowingfor cleaner seed.

4. Alternatively a different herbicide could be deployed in both of thehybrid parents and the mesotrione/HPPD inhibitor is deployed in only themale parent.

5. Other embodiments for deploying herbicide resistant lines includeother traits such as resistance to other classes of herbicides, or othertraits of importance.

Example 5 Seed Production

The herbicide resistance is also used for seed production. As anexample, if it is deployed into the female parent, making it resistant,the herbicide is applied to the seed production field to kill the maleplants before setting seed so that a seed production field is harvestedas a bulk. In addition the purity of the seed may also be verifiedthrough deploying two herbicide resistances with only one in eachparent. Selfed seed is detected and eliminated by applying herbicide putinto the other parent.

Example 6 Control of Broadleaf Weeds and Limited Control of Grasses

The resistance when deployed in a hybrid, by any combination, providesresistance to mesotrione or other HPPD inhibiting herbicides. Thisdeployment results in a new mode of action in rice to control broadleafweeds with some limited control of grasses such as red rice. Furtheroptions or broad spectrum control of weeds is provided by deployment inthe same hybrid another resistance to herbicides providing grass weedcontrol, such as ACCase inhibiting herbicides. Through deployment withother modes of action development of weed resistance is more likely tobe prevented through the use of multiple modes of action.

Example 7 Selection of Herbicide Resistant Rice Using a HerbicideBioassay

Selection of material inheriting mesotrione tolerance is accomplished bya simple herbicide bioassay. A high rate of mesotrione (at least 420 gmai/ha) is applied allowing differentiation of heterozygous individualsfrom homozygous individuals and the tolerance level of the mutation linefrom the inherent tolerance level in the background of some types ofrice. In one example a rate of 105 gm ai/ha is applied followed threeweeks later by a second application of 630 gm ai/ha. In another examplea rate of 420 gm ai/ha is applied in a single application. Yet anotherexample entails applying the herbicide at a rate of 630 gm ai/ha.Herbicide applications are done at the three to four leaf stage ofseedling growth. The ideal situation is to have also planted near orwithin the plants to be selected a set of plants from the originalmutant mesotrione tolerant donor line ML0831266-03093, a row of plantsof the wild-type of the mutation line, P1003, and a row of the lineinvolved as the other parent in the cross. These control lines alloweasy differentiation for inheritance of the tolerance provided by theML0831266-03093 mutant line through comparison of the response in theplants to be selected with the control lines. Only plants that live andare relatively healthy will have inherited and be homozygous for thetolerance level provided by the ML0831266-03093 mutant line (FIG. 18).

Example 8 Production of Rice Resistant to Both HPPD and ACCaseInhibiting Herbicides

The mutant line ML0831266-03093 that is tolerant to mesotrione andlikely other herbicides including HPPD inhibitors, was crossed withmutant line ML0831265-01493 having tolerance to ACCase herbicides andmore specifically “fop” type of ACCase herbicides. In one example theML0831266-03093 plants are the female parent and pollination is by aplant from line ML0831265-01493. In another embodiment the parents arereversed so that ML0831266-03093 serves as the pollinating parent. Theresulting F1 seed are harvested having inherited both mesotrione andACCase herbicide tolerance. The F1 individual carries tolerance to bothherbicides at a partially dominant level so they show some tolerance butnot to the same level as the tolerant parent lines.

The F1 seeds are planted and the resulting plants are allowed toself-pollinate to produce F2 seed making a population segregating fortolerance to both mesotrione and ACCase herbicides. This population isscreened by an herbicide bioassay to identify individuals that haveinherited tolerance from the original mutant line ML0831266-03093 andare homozygous for the resistance. A high rate of mesotrione is appliedallowing differentiation of heterozygous individuals from homozygousindividuals and the tolerance level of the mutation line from thetolerance level in the background of some types of rice including theoriginal line used for mutation to create the ML0831266-03093 line,which was P1003 (FIG. 3).

In one example a rate of 105 gm ai/ha is applied followed three weekslater by a second application of 630 gm ai/ha. In another example a rateof 420 gm ai/ha is applied in a single application. Yet another exampleentails applying the herbicide at a rate of 630 gm ai/ha. Herbicideapplications are done at the three to four leaf stage of seedlinggrowth. The ideal situation is to have also planted near or within theF2 population a set of plants from the original mutant mesotrione donorline ML0831266-03093, a row of plants of the wild-type of the mutationline, P1003, and a row of the line involved as the other parentML0831265-01493. These control lines will allow easy differentiation forinheritance of the tolerance provided by the ML0831266-03093 mutant linethrough comparison of the response in the F2 plants to these controllines. Only plants that live and are relatively healthy will haveinherited and be homozygous for the tolerance level provided by theML0831266-03093 mutant line.

Example 9 A Co-Dominant Marker Assay to Select and Develop ACCase andHPPD Tolerant Rice Lines

A simple co-dominant marker assay is available to select for inheritanceto ACCase herbicides derived from line ML0831265-01493. The marker isdeveloped as a single nucleotide polymorphic marker and detects thecausal mutation at position G2096S (blackgrass number) for ACCasetolerance in line ML0831265-01493. All of the surviving plants followingthe mesotrione bioassay as employed in Example 8 are sampled for tissuecollection, the DNA is extracted by known methods and the samples aretested with the SNP assay. A subset of the surviving plants are thenalso identified as carrying homozygous tolerance to ACCase herbicidesthrough marker assisted selection.

Individuals with tolerance to both mesotrione and ACCase herbicides areselfed to produce F3 families and further selected for other importantagronomic characters. The F3 lines are selfed and purified to derive anew line or variety with dual resistance to mesotrione and ACCaseherbicides. Such lines are highly valuable as the use of both herbicidesprovides more complete and broad-spectrum weed control.

In another embodiment the individuals with tolerance to both mesotrioneand ACCase are used as trait donors in a backcross (BC) breedingprogram. After selecting one individual or a few individuals they areused either as the pollinating parent or the female parent. Another moreelite and desirable line serves as the recurrent parent to which thetraits are transferred.

Following the first cross the F1 plants are crossed again to therecurrent parent. The resulting backcross seed from this cross andongoing crosses to the recurrent parent are tested with either markersor through herbicide bioassays for inheritance of the herbicidetolerance or a combination of markers and bioassays. In the bestsituation markers for the functional mutations are used. Alternativelyan herbicide bioassay for mesotrione is applied to the BC seed orpossibly the BC seed is progeny tested to verify inheritance of thetolerance. Furthermore an herbicide bioassay is used to identifyindividuals that also inherited tolerance to ACCase herbicides. Thisprocess is repeated until the recurrent parent genome is recovered alongwith the two new traits for tolerance to mesotrione and ACCaseherbicides. After the last backcross individuals are selfed to recoverthe dual herbicide tolerances in a homozygous resistant level in atleast one plant.

In yet another embodiment the individuals with resistance to bothmesotrione and ACCase herbicides are crossed to a third line andsubsequently selfed or even crossed with other lines. The resulting newlines and germplasm is tested and evaluated for other agronomicimportant traits. Finally new varieties or male and female lines aredeveloped with tolerance to both mesotrione or other HPPD herbicides andACCase herbicides a combination novel to rice.

Example 10 Mutant Rice ML0831266-03093

The mutant line ML0831266-03093 is demonstrated to carry a hightolerance level to mesotrione herbicide beyond the tolerance foundnaturally in some rice types including the original mutation treatedline P1003. The mutant line is planted in rows or alternatively wholeplots are planted and rows of the unmutated line (P1003) and other typesof rice or whole plots are planted. Mesotrione is applied pre-emergenceor alternatively it is applied post-emergence at the three to four leafstage of the rice plants. Various rates of mesotrione are appliedpre-emergence, pre-emergence followed by post-emergent, or post-emergentwith a single or sequential application. Post-emergent applications areapplied at the 3-4 leaf stage of the rice.

With low rates (105 gm ai/ha) of mesotrione applied both the mutant lineas well as the original unmutated line survive. However, other types ofrice, such as the mutant line with ACCase tolerance ML0831265-01493 andthe associated unmutated line R0146 are killed at these rates ofmesotrione. Applying mesotrione herbicide at higher rates clearly showsnew and novel tolerance level as only the mutant line ML0831266-03093survives while the original unmutated line P1003 and all other testedlines are killed or severally injured. The higher tolerance tomesotrione makes the line ML0831266-03093 of commercial value as boththe tolerance can be controlled or bred into new varieties and it is ofa high enough level to allow commercial weed control in rice with theapplication of mesotrione herbicide and possibly other HPPD inhibitingherbicides (FIG. 19A initial results, FIG. 19B new results after 1 year,and FIG. 22-trial results on various rice lines).

Example 11 Chromosomal Locations of Mutations Related to HPPD InhibitingResistance

The mesotrione tolerant line ML0831266-03093 is tolerant to rates of 420gm ai/ha and even a dual application of mesotrione first at a rate of105 gm ai/ha followed three weeks later by an application of 630 gmai/ha. The line ML0831266-03093 with this high level of tolerance iscrossed with a line highly sensitive to mesotrione one being lineML0831265-01493, which has tolerance to ACCase herbicides. The cross ismade and the resulting F1 seeds are harvested, planted, and allowed toself to produce a F2 population. The F2 population is grown and tissueis collected from individual plants. Each F2 plant and parental linesare tested with a set of 192 SNP markers identified as being polymorphicbetween the two mutant lines ML0831266-03093 and ML0831265-01493. Theset of polymorphic markers was identified as evenly spaced across therice genome after testing both parental lines with a set of 796 SNPmarkers. All 192 markers including two found with linkage to targettraits were selected from the 44 k SNP set described by Zhao et al.2011.

Seedlings of size 3 to 4 leaves are sprayed with mesotrione herbicide.In one set of 89 plants mesotrione is first applied at 105 gm ai/hakilling 23 plants. Both the mutant line control ML0831266-03093 and theunmutated line P1003 survived the herbicide application while theunmutated line R0146 was killed. The surviving plants including someadditional plants making a total of 95 are then sprayed with anothertreatment of mesotrione at a rate of 630 gm ai/ha killing or injuring 67while 28 survived. (FIG. 23) The surviving ratio of plants to thosekilled or injured fits a one quarter ratio with a chi squared value of1.03, well within the expected for a single recessive gene. Another setof F2 individuals of size 78 individuals are sprayed with a singleapplication of mesotrione herbicide at a rate of 420 gm ai/ha resultingin 26 plants surviving also fitting in the expected one quarter ratiofor a single recessive gene with a chi square value of 2.88 (Table 2).

Example 12 Crosses Between Mutant Lines Resistant to DifferentHerbicides

The cross between mutant line ML0831266-03093 (ATCC PTA-13620) carryingtolerance to mesotrione and possibly other HPPD inhibiting herbicideswith mutant line ML0831265-01493 with (ATCC PTA-12933) tolerance toACCase herbicides produced F1 seed inheriting both herbicide tolerances.Following selfing of the F1 plants F2, individuals are selected eitherthrough herbicide bioassays or alternatively with molecular markers. Inthe case of ACCase a functional molecular marker is described such thatthe mutation at position G2096S (based on the black grass numberingsystem) is selected. Furthermore using either markers linked to the QTLson chromosome 1 and chromosome 2 or herbicide bioassays recovery oftolerance to mesotrione and ACCase including other HPPD herbicides orother herbicides.

Individuals selected for tolerance to mesotrione including other HPPDherbicides and possibly other herbicides may be used in a backcrossconversion program or in breeding to develop new varieties and hybridswith a commercial level of tolerance to mesotrione and other herbicides.Selection with either bioassays or the chromosome 1 and chromosome 2QTLs leads to the recovery of the inherent tolerance in P1003 along withthe mutant tolerance for development of a novel variety or hybrid withherbicide tolerance and representing new weed control options in rice.The tolerance level of the mutant line is superior to other lines andallows for various commercial application methods.

The individual plants with tolerance to both herbicides are used inbreeding to develop new varieties and hybrids with tolerance to bothACCase inhibiting herbicides, mesotrione, other HPPD inhibitingherbicides, and other herbicides. The new varieties and hybrids arecommercial products. The commercial products are used commercially forrice production. In the production process both ACCase and mesotrione orother herbicides may be applied to the rice crop to control weeds. Inone example mesotrione or other herbicides are applied preplant tocontrol germinating weeds and provide residual weed control. Followinggermination of the rice crop ACCase herbicides are applied forcontrolling grass weeds. In another example both mesotrione or theequivalent is applied preplant and a second application is made postemergent along with an ACCase herbicide with one or two applications. Inanother example both mesotrione and an ACCase herbicide or otherherbicides including other HPPD herbicides are applied post emergentwith one or two applications. In this manner a new and novel strategy isimplemented to provide full spectrum weed control in rice. In additionthese herbicides have new not previously used in rice modes of action.This strategy therefore has commercial application not only for weedcontrol but as a method to extend the useful life of this strategy andothers through the application of multiple modes of action for weedcontrol.

Example 13 Identification of the Causal Mutation for Tolerance toMesotrione and Other HPPD Herbicides

The mutant line ML0831266-03093 is crossed back to the original line,P1003, used for induction of mutations. The F1 seed is grown and theplants selfed to produce an F2 population segregating for tolerance tomesotrione. Each F2 plant is labeled and a leaf sample is collected forDNA extraction. The F2 plants are then sprayed with mesotrione herbicideat the 3-4 leaf stage at a rate of 630 gmai/ha. Among the surviving setof the least injured twenty are identified and used for DNA extractionto represent individuals that inherited the mesotrione tolerance andpresumably the causal mutation for tolerance. Out of the plants killedby the herbicide application a set of twenty is also identified for DNAextraction to represent individuals that have the wild-type allele atthe causal tolerance locus.

The leaf tissue from the identified individuals is used for DNAextraction and DNA is combined from each set to make a bulk ofindividuals carrying the tolerance and a bulk lacking the tolerance. Inaddition DNA is also extracted from leaf tissue derived from theoriginal line, P1003, used for mutation treatment. These samples areused in next generations sequencing at 30× coverage and compared to therice reference sequence, NIPPONBARE, to the original line used formutation treatment, P1003, and to each other. Using these comparisons itis possible to identify among the group with tolerance a single mutationbeing present across all individuals and thus highly likely to be thecausal mutation for tolerance to mesotrione.

The genome regions suspected to carry the causal mutation are sequencedby Sanger sequencing technology in both the mutant line ML0831266-03093and the original non-mutant line P1003. Following the identification ofreal SNP markers or some other suitable marker is developed and thewhole F2 phenotyped segregating population is tested to identify linkageof the suspected causal mutation to the phenotype.

Other characterizations and processes are also applicable to verify thefunction of the causal mutation. For example the gene containing themutation is identified through comparison to the published full ricesequence and related databases. Furthermore the gene product or enzymecan be isolated and characterized to describe its normal function andfunction with the new mutation especially in relation to its function inthe presence of mesotrione.

Example 14 Double Mutant Resistant to HPPD and ACCase Herbicides

A simple co-dominant marker assay is available to select for inheritanceto ACCase herbicides derived from line ML0831265-01493. The marker isdeveloped as a single nucleotide polymorphic marker and detects thecausal mutation at position G2096S (blackgrass number) for ACCasetolerance in line ML0831265-01493. All of the surviving plants followingthe mesotrione bioassay as employed in example 11 are sampled for tissuecollection, the DNA is extracted by known methods and the samples aretested with the SNP assay. A subset of the surviving plants are thenalso identified as carrying homozygous tolerance to ACCase herbicidesthrough marker assisted selection.

Individuals with tolerance to both mesotrione and ACCase herbicides wereselfed to produce F3 families and further selected for other importantagronomic characters. The F3 lines were selfed and purified to derive anew line or variety with dual resistance to mesotrione and ACCaseherbicides. Such lines are highly valuable as the use of both herbicidesprovides more complete and broad-spectrum weed control. For example linePL1214418M2-73009 (ATCC deposit PTA-121398) and PL1214418M2-80048 (ATCCdeposit PTA-121362) contains tolerance to both HPPD inhibiting herbicidemesotrione and the ACCase inhibiting herbicide fluazifop (see Table 8).Other related lines have also been developed and are highly useful foruse as a new weed control system in rice employing both ACCase and HPPDtypes of herbicide.

In another embodiment the individuals with tolerance to both mesotrioneand ACCase are used as trait donors in a backcross breeding program.After selecting one individual or a few individuals they will be usedeither as the pollinating parent or the female parent. Another moreelite and desirable line serves as the recurrent parent to which thetraits are transferred. Following the first cross the F1 plants arecrossed again to the recurrent parent. The resulting backcross seed fromthis cross and ongoing crosses to the recurrent parent are tested witheither markers or through herbicide bioassays for inheritance of theherbicide tolerance or a combination of markers and bioassays. In thebest situation markers for the functional mutations are used.Alternatively an herbicide bioassay for mesotrione is applied to the BCseed or possibly the BC seed is progeny tested to verify inheritance ofthe tolerance. Furthermore an herbicide bioassay is used to identifyindividuals that also inherited tolerance to ACCase herbicides. Thisprocess is repeated until the recurrent parent genome is recovered alongwith the two new traits for tolerance to mesotrione and ACCaseherbicides. After the last backcross individuals are selfed to recoverthe dual herbicide tolerances in a homozygous resistant level in atleast one plant.

In yet another embodiment the individuals with resistance to bothmesotrione and ACCase herbicides are crossed to a third line andsubsequently selfed or even crossed with other lines. The resulting newlines and germplasm is tested and evaluated for other agronomicimportant traits. Finally new varieties or male and female lines aredeveloped with tolerance to both mesotrione or other HPPD herbicides andACCase herbicides a combination novel to rice.

Example 15 Full Spectrum Weed Control in Rice Based on Dual Resistanceto Both ACCase and HPPD Herbicides

In the production process both ACCase and mesotrione or other herbicidesmay be applied to the rice crop to control weeds. In one examplemesotrione or other herbicides are applied preplant to controlgerminating weeds and provide residual weed control. Followinggermination of the rice crop ACCase herbicides are applied forcontrolling grass weeds. In another example both mesotrione is appliedpreplant and a second application is made post emergent along with anACCase herbicide with one or two applications. In another example bothmesotrione and an ACCase herbicide or other herbicides including otherHPPD herbicides are applied post emergent with one or two application.In this manner a new and novel strategy is implemented to provide fullspectrum weed control in rice. In addition these herbicides have notpreviously been used in rice modes of action. This strategy thereforehas commercial application not only for weed control but as a method toextend the useful life of this strategy and others through theapplication of multiple modes of action for weed control.

Seed Deposits Under Budapest Treaty

Seed deposits by Ricetec AKTIENGESELLSCHAFT were made with the AmericanType Culture Collection (ATCC), 10801 University Boulevard, Manassas,Va. 20110, United States of America. The dates of deposit and the ATCCAccession Numbers are: ML0831266-03093 (PTA-13620, Mar. 19, 2013);ML0831265-01493 (PTA-12933, May 31, 2012); ML0831265-02283 (PTA-13619,Mar. 19, 2013); PL1214418M2-73009 (PTA-121398, Jul. 18, 2014);PL1214418M2-80048 (PTA-121632, Jun. 30, 2014) (see also Table 8). Allrestrictions will be removed upon granting of a patent, and the depositsare intended to meet all of the requirements of 37 C.F.R. §§1.801-1.809,and satisfy the Budapest Treaty requirements. The deposit will bemaintained in the depository for a period of thirty years, or five yearsafter the last request, or for the enforceable life of the patent,whichever is longer, and will be replaced as necessary during thatperiod.

DEFINITIONS

In the description and tables which follow, a number of terms are used.In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

Acetyl-Coenzyme. A carboxylase (ACCase; EC 6.4.1.2) enzymes synthesizemalonyl-CoA as the start of the de novo fatty acid synthesis pathway inplant chloroplasts. ACCase in grass chloroplasts is a multifunctional,nuclear-genome-encoded, very large, single polypeptide, transported intothe plastid via an N-terminal transit peptide. The active form in grasschloroplasts is a homodimeric protein.

ACCase enzymes in grasses are inhibited by three classes of herbicidalactive ingredients. The two most prevalent classes arearyloxyphenoxypropanoates (“FOPs”) and cyclohexanediones (“DIMs”). Inaddition to these two classes, a third class phenylpyrazolines (“DENs”)has been described.

Certain mutations in the carboxyl transferase region of the ACCaseenzyme results in grasses becoming resistant to ACCase herbicides. Inthe weed Black-Grass at least five mutations have been described whichprovide resistance to FOP or DIM class of ACCase herbicides. Somemutations rendering ACCase enzymes resistant to these herbicides may beassociated with decreased fitness.

Allele. Allele is any one of many alternative forms of a gene, all ofwhich generally relate to one trait or characteristic. In a diploid cellor organism, the two alleles of a given gene occupy corresponding locion a pair of homologous chromosomes.

Backcrossing. Process of crossing a hybrid progeny to one of theparents, for example, a first generation hybrid F1 with one of theparental genotypes of the F1 hybrid.

Blend. Physically mixing rice seeds of a rice hybrid with seeds of one,two, three, four or more of another rice hybrid, rice variety or riceinbred to produce a crop containing the characteristics of all of therice seeds and plants in this blend.

Cell. Cell as used herein includes a plant cell, whether isolated, intissue culture or incorporated in a plant or plant part.

Cultivar. Variety or strain persisting under cultivation.

Embryo. The embryo is the small plant contained within a mature seed.

Essentially all the physiological and morphological characteristics. Aplant having essentially all the physiological and morphologicalcharacteristics of the hybrid or cultivar, except for thecharacteristics derived from the converted gene.

Grain Yield. Weight of grain harvested from a given area. Grain yieldcould also be determined indirectly by multiplying the number ofpanicles per area, by the number of grains per panicle, and by grainweight.

Injury to Plant. Is defined by comparing a test plant to controls andfinding the test plant is not same height; an abnormal color, e.g.yellow not green; a usual leaf shape, curled, fewer tillers.

Locus. A locus is a position on a chromosome occupied by a DNA sequence;it confers one or more traits such as, for example, male sterility,herbicide tolerance, insect resistance, disease resistance, waxy starch,modified fatty acid metabolism, modified phytic acid metabolism,modified carbohydrate metabolism and modified protein metabolism. Thetrait may be, for example, conferred by a naturally occurring geneintroduced into the genome of the variety by backcrossing, a natural orinduced mutation, or a transgene introduced through genetictransformation techniques. A locus may comprise one or more allelesintegrated at a single chromosomal location.

Induced. As used herein, the term induced means genetic resistanceappeared after treatment with mutagen.

Non-induced. As used herein, the term non-induced means geneticresistance not known to be induced; is at different location in thegenome, than induced resistance.

Plant. As used herein, the term “plant” includes reference to animmature or mature whole plant, including a plant from which seed orgrain or anthers have been removed. Seed or embryo that will produce theplant is also considered to be the plant.

Plant Part. As used herein, the term “plant part” (or a rice plant, or apart thereof) includes protoplasts, leaves, stems, roots, root tips,anthers, seed, grain, embryo, pollen, ovules, cotyledon, hypocotyl,glumes, panicles, flower, shoot, tissue, cells, meristematic cells andthe like.

Quantitative Trait Loci (QTL). Genetic loci that controls to some degreenumerically measurable traits that are usually continuously distributed.

Regeneration. Regeneration refers to the development of a plant fromtissue culture.

Resistance/Resistant¹. The inherited ability of a plant to survive andreproduce following exposure to a dose of herbicide normally lethal tothe wild type resistance may be naturally occurring or induced by suchtechniques as genetic engineering or selection of variants produced bytissue culture or mutagenesis. ¹ Weed Science Society of America, WeedTechnology, vol. 12, issue 4 (October-December, 1998, p. 789)

Single Gene Converted (Conversion). Single gene converted (conversion)includes plants developed by a plant breeding technique calledbackcrossing wherein essentially all of the desired morphological andphysiological characteristics of an inbred are recovered, whileretaining a single gene transferred into the inbred via crossing andbackcrossing. The term can also refer to the introduction of a singlegene through genetic engineering techniques known in the art.

Tolerance/Tolerant. The inherent ability of a species to survive andreproduce after herbicide treatment implies that there was no selectionor generic manipulation to make the plant tolerant.

Resistance/tolerance are used somewhat interchangeably herein; for aspecific rice plant genotype information is provided on the herbicideapplied, the strength of the herbicide, and the response of the plant.

Sequence listing of HPPD gene and proteinLOC_Os02g07160 sequence informationGenomic sequence length: 2760 nucleotides CDS length: 1341 nucleotidesProtein length: 446 amino acidsPutative Function: glyoxalase family protein, putative, expressedGenomic Sequence >LOC_Os02g07160 (SEQ ID NO: 1)ACGCCGCCACTGTCATCCACTCCCCCACACCCCACGACGCGCCACGCCACGCCGCGCCGCGCCGCGCCATGCCTCCCACTCCCACCCCCACCGCCACCACCGGCGCCGTCTCGGCCGCTGCGGCGGCGGGGGAGAACGCGGGGTTCCGCCTCGTCGGGCACCGCCGCTTCGTCCGCGCCAACCCGCGGAGCGACCGGTTCCAGGCGCTCGCGTTCCACCACGTCGAGCTCTGGTGCGCCGACGCCGCGTCCGCCGCGGGCCGGTTCGCCTTCGCCCTGGGCGCGCCGCTCGCCGCCAGGTCCGACCTCTCCACGGGGAACTCCGCGCACGCCTCCCTCCTCCTCCGCTCCGCCTCCGTCGCGTTCCTCTTCACCGCCCCCTACGGCGGCGACCACGGCGTCGGCGCGGACGCGGCCACCACCGCCTCCATCCCTTCCTTCTCCCCAGGCGCCGCGCGGAGGTTCGCCGCGGACCACGGCCTCGCGGTGCACGCCGTGGCGCTGCGCGTCGCCGACGCGGCCGACGCCTTCCGCGCCAGCGTCGCGGCCGGTGCGCGCCCGGCGTTCCAGCCCGCCGACCTCGGCGGTGGCTTCGGCCTCGCGGAGGTGGAGCTCTACGGCGACGTCGTGCTCCGCTTCGTCAGCCACCCGGACGGCGCCGACGCGCCCTTCCTCCCGGGTTTCGAGGGCGTCAGCAACCCGGGCGCCGTGGACTACGGCCTCCGCCGGTTCGACCACGTCGTCGGCAACGTGCCGGAGCTCGCTCCGGTAGCCGCGTACATCTCCGGGTTCACCGGGTTCCACGAGTTCGCCGAGTTCACCGCCGAGGACGTGGGCACCGCCGAGAGCGGCCTCAACTCGGTGGTGCTCGCCAACAACGCGGAGACCGTGCTGCTGCCGCTCAACGAGCCGGTGCACGGCACCAAGCGGCGGAGCCAGATACAGACGTACCTGGACCACCACGGCGGCCCGGGGGTGCAGCACATCGCGCTGGCCAGCGACGACGTGCTCGGGACGCTGAGGGAGATGCGGGCGCGCTCCGCCATGGGCGGCTTCGAGTTCTTGGCGCCGCCGCCGCCCAACTACTACGACGGCGTGCGGCGGCGCGCCGGGGACGTGCTCTCGGAGGAGCAGATCAACGAGTGCCAGGAGCTCGGGGTGCTCGTGGACAGGGATGACCAGGGGGTGTTGCTCCAGATCTTCACCAAGCCAGTAGGAGACAGGTAAAATCCTCACCTCTTTCATGATGAAAATGGCTTATGAATTCAGATTTGCAGTTATTTGTTGGCACATAGCATCGATTAGGCGCAGAAAGGTGTCAAGCATTATGAAATTAATCCAGAATGCTTGAATAATACAGTATAATATATGATAGTGAGCTCTGTGATACTCCATGGATACTCTTTATGTGTCTCCATGAATCCATGATGCGCCTTTCTGAAGATTGTGACACTAGAAAGGGAATAAAGCTGAATGTGCATAGGAAAAAAATGAAAAGCCAATGTGTGTCTGTTTATGCCTTCTTGCAAGCATATCCCAGTTCCTTTTTGCCGGCATGTTGTAATGCAGATAGCCAGCCACATATAGCTACTTAATTAGTGAGTACTCCCTCTCACAATGTAAGTCATTCTAGTATTTTCCACATTCATATTGATGCTAATCTATCTAGATTCATTAGCATCAATATGAATATGGGAAATACTAGAATGACTTACATTGTGAAACGGAGGAAGTATTACTTACTACATCTAAGGTCCATGGATTCCTTTTTTTACAAAAGAAAGAAAGAATCTTATGGCAACTCCATCAGCATAAACCAGCAATGCTGCTGGGAACAACTTAAACTTTAGGTTCAGGAGGTTGTAATTGTCTTTAAGCTTAATAGTCTGATTCAGTCAGTATTCTAATTTCTGCTGCATCTTTGCTATTGTTATTTCCTCTCTGTGACTCCAAATCTAACTGGATCAGCTATTTCACTCAGGCCAACCTTTTTCTTGGAGATGATACAAAGGATTGGGTGCATGGAGAAGGATGAGAGTGGGCAGGAGTACCAGAAGGGCGGCTGCGGCGGGTTTGGGAAGGGCAACTTCTCGGAGCTGTTCAAGTCCATTGAGGAGTATGAGAAATCCCTTGAAGCCAAGCAAGCCCCTACAGTTCAAGGATCCTAGGTAGGAACTGGAGGCCTGGAGCAACAGATGTAACCAGTGTATTTGTATTATGGAGCAGAAGAAAAAAGATGTGCTTTCACTGCTTTGTGATATGTGTCATGCAAGTTGATGTTGTAATTTGTGGAAGCTGAAGACAAATGATGGTACAATCACTGTAATAGATAATAGACATGGATCACATACAAGAATGTAACCTAGTGTTGGCATTGCTGCTGTACAATCTTGCTTGGAAATAAAATAATAATCAACCTGGAGAAAGAATGTAACCTACTGTTGGCATTGCTGATGTACAATCTTGCTTGGAAATAAAATAAGAATCAACCAAGAGAATCTGTCCTTGTGATGCTTGTGATCTTCTGGTGTCTTTTTATTTAACAGAATGTAGTGGTCCTCTGCTGCCTCCAACCGTCCAGGGTAAAAGTGTAAACCGTGGGCTGAGTTACAGCGAATTGCAGTTAGCAATCTGCAAGAGACAGGGGATGAACAGAGTAAGGTCAATAGTTCAGTGTATGACATGATCATCTTGTTTCGTGGCCTTAAATGGCAAGAAAATGGGCTTGTCAGATCTCAAAGAACTCCTATATGTTAAAAGGCDS (SEQ ID NO: 2) >LOC_Os02g07160.1 (SEQ ID NO: 2)ATGCCTCCCACTCCCACCCCCACCGCCACCACCGGCGCCGTCTCGGCCGCTGCGGCGGCGGGGGAGAACGCGGGGTTCCGCCTCGTCGGGCACCGCCGCTTCGTCCGCGCCAACCCGCGGAGCGACCGGTTCCAGGCGCTCGCGTTCCACCACGTCGAGCTCTGGTGCGCCGACGCCGCGTCCGCCGCGGGCCGGTTCGCCTTCGCCCTGGGCGCGCCGCTCGCCGCCAGGTCCGACCTCTCCACGGGGAACTCCGCGCACGCCTCCCTCCTCCTCCGCTCCGCCTCCGTCGCGTTCCTCTTCACCGCCCCCTACGGCGGCGACCACGGCGTCGGCGCGGACGCGGCCACCACCGCCTCCATCCCTTCCTTCTCCCCAGGCGCCGCGCGGAGGTTCGCCGCGGACCACGGCCTCGCGGTGCACGCCGTGGCGCTGCGCGTCGCCGACGCGGCCGACGCCTTCCGCGCCAGCGTCGCGGCCGGTGCGCGCCCGGCGTTCCAGCCCGCCGACCTCGGCGGTGGCTTCGGCCTCGCGGAGGTGGAGCTCTACGGCGACGTCGTGCTCCGCTTCGTCAGCCACCCGGACGGCGCCGACGCGCCCTTCCTCCCGGGTTTCGAGGGCGTCAGCAACCCGGGCGCCGTGGACTACGGCCTCCGCCGGTTCGACCACGTCGTCGGCAACGTGCCGGAGCTCGCTCCGGTAGCCGCGTACATCTCCGGGTTCACCGGGTTCCACGAGTTCGCCGAGTTCACCGCCGAGGACGTGGGCACCGCCGAGAGCGGCCTCAACTCGGTGGTGCTCGCCAACAACGCGGAGACCGTGCTGCTGCCGCTCAACGAGCCGGTGCACGGCACCAAGCGGCGGAGCCAGATACAGACGTACCTGGACCACCACGGCGGCCCGGGGGTGCAGCACATCGCGCTGGCCAGCGACGACGTGCTCGGGACGCTGAGGGAGATGCGGGCGCGCTCCGCCATGGGCGGCTTCGAGTTCTTGGCGCCGCCGCCGCCCAACTACTACGACGGCGTGCGGCGGCGCGCCGGGGACGTGCTCTCGGAGGAGCAGATCAACGAGTGCCAGGAGCTCGGGGTGCTCGTGGACAGGGATGACCAGGGGGTGTTGCTCCAGATCTTCACCAAGCCAGTAGGAGACAGGCCAACCTTTTTCTTGGAGATGATACAAAGGATTGGGTGCATGGAGAAGGATGAGAGTGGGCAGGAGTACCAGAAGGGCGGCTGCGGCGGGTTTGGGAAGGGCAACTTCTCGGAGCTGTTCAAGTCCATTGAGGAGTATGAGAAATCCCTTGAAGCCAAGCAAGCCCCTACAGTTCAAGGATCCTAG Protein (SEQ ID NO: 3) >LOC_Os02g07160.1MPPTPTPTATTGAVSAAAAAGENAGFRLVGHRRFVRANPRSDRFQALAFHHVELWCADAASAAGRFAFALGAPLAARSDLSTGNSAHASLLLRSASVAFLFTAPYGGDHGVGADAATTASIPSFSPGAARRFAADHGLAVHAVALRVADAADAFRASVAAGARPAFQPADLGGGFGLAEVELYGDVVLRFVSHPDGADAPFLPGFEGVSNPGAVDYGLRRFDHVVGNVPELAPVAAYISGFTGFHEFAEFTAEDVGTAESGLNSVVLANNAETVLLPLNEPVHGTKRRSQIQTYLDHHGGPGVQHIALASDDVLGTLREMRARSAMGGFEFLAPPPPNYYDGVRRRAGDVLSEEQINECQELGVLVDRDDQGVLLQIFTKPVGDRPTFFLEMIQRIGCMEKDESGQEYQKGGCGGFGKGNFSELFKSIEEYEKSLEAKQAPTVQGS*

TABLE 1 Phenotypic characteristics of a mutant rice line showingresistance to mesotrione, with comparison to the original unmutatedparent line. Days to 50% Plant Plant Sheath Designation Heading HeightType Pubesence Color Awns TKW, g yield/plant Unmutated 85 96.5 erectglaborous purple None 23.15 N/A parent line inside sheath ML0831266- 8294 erect glaborous purple None 24.55 4.5 03093F2 inside sheath

TABLE 2 Numbers of tolerant plants after three different herbicidebioassays applied to an F2 individuals derived from a cross ofML0831266-03093 with tolerance to mesotrione and ML0831265-01493 withtolerance to ACCase herbicides. One application of mesotrione at 105gai/ha Single Single followed by application of application of secondmesotrione at mesotrione at application of 105 gai/ha 420 gai/ha 630gai/ha Parameters Tolerant Susceptible Tolerant Susceptible TolerantSusceptible Observed (o) 64 23 26 52 28 67 Expected (e) 21.75 65.25 19.558.5 23.75 71.25 Deviation (o − e) 42.25 −42.25 6.5 −6.5 4.25 −4.25Deviation (o − e)² 1785.06 1785.06 42.25 42.25 18.06 18.06 (o − e)²/e 8227.35 2.166 0.722 0.76 0.253 Chi-Square Value 109.35 2.88 1.013 Degreesof 1 1 1 freedom (df) Probability value 0.08919242 0.31393809 CriticalChi- 3.84 3.84 3.84 Square value at p = 0.05

TABLE 3 Markers used in QTL analysis SEQ ID MARKER NO: WG-id11001864 8WG-id11002275 9 WG-id11003701 10 WG-id11007323 11 WG-wd10001341 12WG-wd11001701 13 WG-wd2002275 14 WG-wd7000143 15 BG-id10001133 16BG-id10003147 17 BG-id10004614 18 BG-id1001716 19 BG-id1012406 20BG-id1015060 21 BG-id1020809 22 BG-id1026723 23 BG-id11000280 24BG-id11000643 25 BG-id11001000 26 BG-id11003263 27 BG-id11005541 28BG-id11011578 29 BG-id12001413 30 BG-id12003453 31 BG-id12006669 32BG-id12010130 33 BG-id2000100 34 BG-id2001406 35 BG-id2002159 36BG-id2004662 37 BG-id2006793 38 BG-id2008132 39 BG-id2009032 40BG-id2010498 41 BG-id2012278 42 BG-id2013398 43 BG-id3002278 44BG-id3006415 45 BG-id3007343 46 BG-id3008063 47 BG-id3008702 48BG-id3011050 49 BG-id3011406 50 BG-id3015075 51 BG-id4001244 52BG-id4002084 53 BG-id4004010 54 BG-id4010543 55 BG-id4012206 56BG-id5003430 57 BG-id5004121 58 BG-id5011704 59 BG-id5014703 60BG-id6007975 61 BG-id6011524 62 BG-id6016683 63 BG-id6016941 64BG-id7006069 65 BG-id8000032 66 BG-id8004971 67 BG-id8006271 68BG-id9003596 69 BG-ud11001609 70 BG-ud7000168 71 BG-ud7000468 72BG-ud7001467 73 BG-ud9000404 74 BG-ud9000939 75 BG-wd12000096 76BG-wd5002107 77 BG-wd7000537 78 BG-wd8000300 79 WG-id10000057 80WG-id1000027 81 WG-id10000678 82 WG-id10005716 83 WG-id10006397 84WG-id10006890 85 WG-id10007362 86 WG-id1000987 87 WG-id1002788 88WG-id1003490 89 WG-id1004858 90 WG-id1005915 91 WG-id1006413 92WG-id1007758 93 WG-id1008433 94 WG-id1011077 95 WG-id1013249 96WG-id1015747 97 WG-id1019114 98 WG-id1022207 99 WG-id1023338 100WG-id12004473 101 WG-id12005677 102 WG-id12007189 103 WG-id12008113 104WG-id12009381 105 WG-id2000711 106 WG-id2003035 107 WG-id2003988 108WG-id2005453 109 WG-id2005879 110 WG-id2007502 111 WG-id2011561 112WG-id2011986 113 WG-id2014452 114 WG-id2015344 115 WG-id2016104 116WG-id3000020 117 WG-id3003557 118 WG-id3003855 119 WG-id3004338 120WG-id3005216 121 WG-id3005783 122 WG-id3009997 123 WG-id3010769 124WG-id3013945 125 WG-id3016222 126 WG-id3017628 127 WG-id3018382 128WG-id4000023 129 WG-id4001471 130 WG-id4002895 131 WG-id4004798 132WG-id4005527 133 WG-id4005882 134 WG-id4006725 135 WG-id4007645 136WG-id4008100 137 WG-id4008430 138 WG-id4008947 139 WG-id4009312 140WG-id4009705 141 WG-id4011039 142 WG-id4011619 143 WG-id4011820 144WG-id5001055 145 WG-id5002055 146 WG-id5002453 147 WG-id5002782 148WG-id5004697 149 WG-id5006824 150 WG-id5007247 151 WG-id5007583 152WG-id5008807 153 WG-id5009334 154 WG-id5010535 155 WG-id6000075 156WG-id6001960 157 WG-id6002888 158 WG-id6003335 159 WG-id6004012 160WG-id6004657 161 WG-id6005348 162 WG-id6007016 163 WG-id6010853 164WG-id6012703 165 WG-id6014165 166 WG-id6016119 167 WG-id7000480 168WG-id7001929 169 WG-id7002851 170 WG-id7003936 171 WG-id7004491 172WG-id8000555 173 WG-id8001575 174 WG-id8002235 175 WG-id8005634 176WG-id8006703 177 WG-id8007014 178 WG-id8007344 179 WG-id8007751 180WG-id9000056 181 WG-id9002563 182 WG-id9002755 183 WG-id9005502 184WG-id9006187 185 WG-id9006850 186 WG-id9007344 187 WG-ud1001267 188WG-ud7000348 189 WG-ud7001018 190 WG-ud7002024 191 WG-id1028225 192WG-id11000006 193 WG-id11007850 194 WG-id11008114 195 WG-id11009132 196WG-id12002544 197 WG-id11006215 198 WG-id11002912 199

TABLE 4  SNP markers Chromo- SEQ ID ID some upstream sequence Alleledownstream sequence NO: WG- 1 ATGCCCACGGCGGCGGCGGCGGAGGA C/TGTCGGAAATGCCTGCCACGGGCTGTTCC 4 id1002788 GGAGGAGGAGGAGGAGCTAAGGAGCGCGCAGGTATTGAGAAATGAGCGCTGAG GCGCGGTACGTCGCCGGTGCTGTTCTGCTTCCTGACGCGTTTAAATCCACTGATTA TTTGTAGCCGCTGCTGTCCT GCTGAGTTCCCTTCCAA WG-1 GAGAGTGGAGGAGGAGGACGAGTGGA A/G CAAGCAAAGGAAGCAAGCAAAAGAAA 5 id1003490GGTGGAGGTGGCGCGCGGCTGCGCGG AAAAGCCCGGGAATTTACCTGGCGGGATGCGCTTCTTTTTTTTTTCTTTTTTTGTTC ATGCCCTACTTGGCAGCGCCGCCCGTCTCCGCCGCAACCAAAGGAG CTCTCCACAAACGCCCTGC BG- 2TTCTATCTCAAGGCGGCAATAGAATCAT C/T GCCAAATGTCTGATGAATTGCTCTTGCT 6id2004662 AGATGCTAGAGTCCAGAAGAAGGCCAA CTGATGTTGAGCCCGATGAAGTTGTTAGAGACTTGAAATTTTCAGTTGAGAATGAG CTGCTGAGGACATGATCGGTACCACCTACAATCCAAGGTGATGCT TATTGACAACCCTGAT WG- 2 TCAGTGTTCACGGACCCTACATGGAGTTA/G TATCAATCGATCAATCACCAATCGGATG 7 id2003988CTCCTAAGTTCAACTACAAGAGACATAG GTACCAAATCCAAAACAACAGTTGGGGCCCATAGGGTAATGCCCTCACTTTCCAG AAAACTGATCCTACCAACCCAGCTCAACCTCTTTAACTATGGAG TAATTTTGCAGTGCTAC

TABLE 5  Unknown ACCase mutation-QTL start and end SNP marker sequencesSEQ Chromo- ID ID some Upstream sequence Allele Downstream sequence NO:id1019752 1 Ataaagatgaggtgtttgatgaattaaaggccgca g/tacagcaactgatgcagctgctgcgaaagcccata 206gggttgaagaggccatgtagctttacagatatttc ttaggcgccagcttcatccagatgtctgttcccacagtgaaaatgctttgcttcttgaatttga ggacaagaatacttctggtcatgaactttttgid1025754 1 tcacatgatctgcaactgtcaacagtcttaccgga t/cgcatgatgtgtctatactctatactgcaaagatg 207attggattctgaaggtggatactccacctgtccac aatactaacaagtttttcttgggcttaaaagaagcataagtccttatttgtcagaggttacaat aaaaactaggaacagcctcactagtttgctag

TABLE 6 List of mutations identified in Quizalofop mutant. These mutations were identified usingMutMap method (whole genome sequencing) SEQ ID ID ChromosomeUpstream sequence Allele Downstream sequence NO: Chr1QMM32785958 1tgtttcaagtttggttgctgaaaaacgtacggacaaa g/acttcggtggtttgccaagaacatgaaacctggggataa 208ccatgaactaacttgctattttgctccacatgggtttggatttttttcgactttgctaatgctggtcgtctggat gtctcgaagcaaccaggaattgatcataggaacagaacaggtattgctac Chr1QMM33147227 1agtctaaatgggctgcactttgattgggctgggttca a/ggggtaaacggtgtgcggacgtgagacgagaaaagcatg 209tatgagattaggggaaaaaacacgaacattccagtaaagagaaaacgatctgtgtgcatgcatagggctggacga aatggggagtgtgaactgtgaagaagaaagctcgtgactcgttagctcgc Chr1QMM33201871 1tagggcttctgatagccctccatctgtccgtcctttg t/ctcttcaactcgattggaacagcaggctccgtgtatgtg 210cccgtttgcttcttggcctaaaccaccgaaaaggtggtaactatggctgtgtttagatctaaagtttagattcaa gtccgttttgctggacgcctggaataagtatagatttaaacttcagtcat Chr1QMM34374172 1tgctgattctcaggctgattctacttggttggtagaa a/ggagaaaactcgctacactttataggaaatgaactactc 211aatctacttatccaggaacaagcgtagggtaacttttttctaccgacggatatcctaaaggctatcctctggtac cctttttttctcagctatgtgaaaagctcggccttacgctaactcaaaac Chr1QMM34482064 1cgatcccagggaggttgtggaagtgcttctcatgacc a/gacctgaaggctggtgccatcatgttggccagtaatctg 212ttcgaggcactgcggcgtaggcatcttcaattggatgagggctaacattgggaagaggattggagctgtccctgg agacacaagagactagcaaacgtgcaagttgaagtaggggatattttcta Chr1QMM34680949 1aattccaatttcatcccatttgtcccattccctcctg a/gccctccatcagctgaaaaattaccagaaacaaaatatt 213attactttgccaagaaaaataagcctgtggagaattccatctggaaatctgggaaattttcagaacagctcacag atcagatgcaggaatagtgccagaaggtgatgggcagtcaggcagcaaac Chr1QMM34976760 1gttttggttgctattaatcgattgagcaagtagggga t/ccgattacaccgttgtgttcgtaataattaaatctttac 214aatattcctatcatctatgcttcaaataaagttttctaacaagatctcacatgattatattttgatgaaaaatca cttaaattactcatccgatttacaatcaaattacttttatgatatgtcta Chr1QMM35498447 1gtcccgcctggtgacgatttccatgggcattgcgccg a/gagtacacgcgaagactgtaggtagaggtgcttttcccg 215actgactgtgtcggcagcatgcatcgtctcgggcgttcgaaaagtggcagtagcggcggttggacagtaaccttc caacgtgtggaggggacgacgtataagtgtatggttgtgtgttcatctca Chr1QMM35779866 1tgcttgtgcgttcactgttcagagaagctggttatcc a/gtcacccagcacaattgactggctgagtgttgcattaag 216tccctgataagaacagccgggaggtcagtgtgctatgcaaatctggaccggatttgagggaattttctcgcgcag gttttgtttagttctggaatgatccatggagatctatgataaatctcgta Chr1QMM36160202 1ctgcttcgccaacggcctcgaggcgaggctggcaggt a/gcgtaccagctttatttggcagcttgcccatttaagaag 217actggtagtcaaatttacaagaactacacgataactcatctctcattactttgccaatcaaacaattttgaatgc ggcttccatgcactgatgtgctgaaatgtggagaaggccaagaaagttca Chr1QMM36386713 1tcacattctggttgttgagggtccactgataccttta a/gcacagcagcagtacatgcaatgcaagatgcccagatga 218cctgttgcagtttattgttttaaataatccaatcaaatgagaataaaggcaatggcaaaaatataagtgctagtt cttttgtttgagcttattgctgaatactatttcaaaataacaaacagaca Chr1QMM36447011 1tatgatgatgcttattatagcctaaggtatgtacttt a/gccactctacttatgagaagccgggccaccacattcata 219taagatttagttcgaagtaatgcccatctggcaagttcattacagccagaaacaacaaatccaggaagttaatac aattccagcattaacgtgttctaaaagtgattaagaatgcatcaaacaag Chr1QMM36747244 1tcgatatgttgggttttttctctttactagtagcatg a/gctccgaggacgaggaggaggacgactaatttggcagct 220ccatctagtgtgcatcttacgtagtggaatattatcgcagctcacctgcacggctgcactgtgctgtgcccggtg ggcacccaatattcggctcgcacaaaggcgaagccatttcacccgcgggc Chr1QMM28257622 1tctgaaaacgcatggccgaaataagatgcaagaacac a/gctccgaggacgaggaggaggacgactaatttggcagct 221ctgcaaaataatctcaggatcagtccaggtacgcatttgctgcttgtaattctctgaatttctgaatgaaagaaa ctctactgttatctactgaaccagagagaaaagaaaagaagcgaaactgg Chr1QMM29278751 1cttaaattctcatgttttattcccgttgcaacgaacg a/ggtgtggcgcctacgtgatcgttggtttgtttcgcttgt 222gtcatttcttttagtgtccataaatagctataagaggttgggcatacagctatgagaactttgttgggggcccat catcgatcatcgcagcaagccgactgactacttatcatcatgtgtctaat Chr1QMM29340100 1gaactaaacacacccggaatgtgatggatccgaatct a/gaaaaacataggaataagaatcctatgtgaattggtact 223gctgtagttgatactgtgaatgtaacttgtaggcctcgttcatccctttgatttgtaggaattgaacaaaggaaa atttgattttctagaaaaaaatggagagcatggggaaaaaaatcctatga Chr1QMM29875869 1agcgccacgccgcggccagcgccgtggtgttctccgg a/gatccggcggctccacagggccgtcggcaacgcggtcgt 224gtggcatcgcttgagctacatcaccaccgatggccaccgacgacaagtacttggtcttcgggaccggctccaccc ttaaagtccgttgagctcgatcgccaacctgatcaacgcgctggtgtacg Chr1QMM30173016 1acgggcggtaccagctacctgtcacagacatgtgggc a/gtccacgggcgcaccgagcgtccaccgccccccgcgata 225ccagcttaacgctaacgcgctgatggccccacatggctccgggggtttaggcgatatttagccacgagaggggga agcgacctgccccctctgtccctccgggggagagtaggagaccgacgctt Chr1QMM30198493 1agaaagacttgctttagctttattgtttcttttccat a/gcacttcaaaagggatgaaggaaagaaggctgtcatatc 226attctatattcttgaaaaggactgcaaagctcttctaaattactcaatcatgaccagatcatcgatctgatgcag gtatatgcaattagctgctttggaagttaaaaatttcattaattttgcca

TABLE 7 List of mutations identified in Mesotrione mutant. Mutations wereidentified through MutMap method (whole genome sequencing). SEQ ID IDChromosome Upstream sequence Allele Downstream sequence NO:Chr1CMM3349975 1 atcgatgtaattagtgatgtcaatcaatggtccaga a/gTacagcatattccaaggaatctgtgtgcttcctat 227tggcatttggagtcttcgagccttatctttagtgtc caggttttgctggaatggaaaattgtgctggctcaacttgcattttcagttccaaatgaacatctggaaga gttttgctgttccccctacttttcaataaatcaggaggcttggatatctttggaatttcattgataggatg ttcaccttcttttcgcagaccagttgaattttgtaaaaatctcttgccgcatgaagcctgacggagtttcc tattctctttacaagcagatgctgaagagtggcctgttagccaaagtggcatcag aataaattatttggtgaaacctcta Chr1CMM3568176 1Taaccttatagtgggacaaggactgaaaagcagttc a/gaaatagattttctacaagcacatgatcattggtgg 228ctcttgctttaacccagagagggtcaacatttttct gcactgcccacagataaaactagctagctctcggcccctgtaagttccaatgctccacaatatttgtatca agtcttacccatgggaaacagtaggatctatcgaaagtgttttgaggttccaggatatgcttcagaatcca aaatcaagcagctgctttgatataactttccaggcttgctccccatctcaatagaatgcataagctgaaaa agtatagaaatctggcaaagagattagcagctcacggcaagaaaaagtggaataa ctcagaagtgaaatttggagggcaa Chr1CMM4052710 1ccaagcggagacatcgcttccatgagcaattcacgg a/ctgcagcagtcagctcatcagttcttgtaagactca 229 tttttcactagatctctcaccctgatcaacacagattgtgcagccactaagttact ctttctctgatccattctggaccagcctctaatgcgtatactgcattgctatggtgataatttaagggaat tagagcgccaggcgctgcccgatgatggaagcacaggccctggtaaaagattcaagtggatgcgggaaatt ataggtatgttatcttgcactttgagcagctgtgcacatgaaactgaaagaactcaatattgactcttaca tgaagaccgtcagcttcgtttgggtgggcaattcagcattttcaagcctaagcagag Chr1CMM4203161 1tttaaatcaaatcttaaaaatataaatcataaataa t/ctttataagtatggagggagtatccatttcacatat 230ctatcaagttgttgagtttaaaaatataaaaattat acttatggtcttgtttacatcccaacaaattttagataaatatatttgtcttgaaaaatactttcataaaa ccaaaaacatcacatcaaatatttagccacatgtagtatacatatatcactttttaataaatatttttata taggacattaaatataaaaaaacaattacacagttaaaacaagaagtcaaagttatgttttagagaccgcg tgcatgtaaattgcgagacgaatcttttgagcctatctctgttctaaacgacttc attacgccattatttgacaatgtgg Chr1CMM4388604 1cggaactatgactaactcctctccgtaagcttcttt t/cttccagtgttactcaaaatctagctactggaaaca 231gtaatatgtattgctgctgtacttggtctcattatc ataccaatattatagaacaaacagctgatgttatctccttacagatatatatacattttttgcagggtata acaaaaatagacaatagtatgagttcaccacggattccacttcatcttctccgtgacattgagactagggt gagaataacagaaaggaatggtataagggacaacattggctcctggccgtggagtcagagagtcagtgtaa ccaccccatcattccatcaacatttgcaactctttagctgacggagaatatgcac gagtctaatggcgaaggcgtacata Chr1CMM4425961 1caactactgacaacaagtgccatgtctaaatttctg t/ccaacttttaaaaatgtgggaacaatcaaaccatat 232aacatgcacacaacacacaaatgatgaatatggtga gcttgagatatacccacaaagccatcggcggccgcaaccgcaattagcattagaaagttttaactctagaa ttacagcagagtacaccctcatcttgcacgcctccatcaatttccaagttgtaatccccatactcccaacc gaagaagcaggagccgtgatcaacacgagcatcttcagaagggaaaaaaaacaactccaaaaccc gtcctgctccaccccacacagtgacgagtctagggcacgacgaaggcgcgctcctaaacc Chr1CMM4454432 1cttcctgtcgtgagtgactgggtggtgggctcaatc t/ctagtgctgttctgaacctcttgcgcatacattaac 233ggcctggcccgatacgatgcaagcgcgtggctgggc atgttttatctaatctaataaacatgattaaatttaggagatcggacggtgctgattgttggggcgacgtg agcgtttgcttttacagtagtagaaatatgaaattgccgcgtgggcgaatgaatagtgaacagtaccgacg gaacaatggttagtctgaggaatcataagcctatgtgaggtttataggattttatatgactaggggtgaac atctagctggagtcttctccggtttaagctaccaagttggatagaagggaatgtg ttgaaacatattaattgatgcctga Chr1CMM4496531 1aatgtgctcaacttcatatatatgtgtgttgagcac a/gaatattcaactttactcaatgttttgtttaacaag 234atagctcatatataagatcaatggttagatcaatgg ttccttttggtcacttgccaatttttctagatcattttttgggttagatcaatggttgagcacatagctca acagtacaatctattgatcacaattcacattgaattatatatgtgtgctcaacggctcacacacaacttca aactaggtcaagccattctgtacatgcccatgcattatatatgtacccaaaaaagcactattagatcaatg gaacttactgtactaatattatcttagattaatttgttataattgtttcaccacg atcctgaaacttatagtcatatgtg Chr1CMM3714792_CA-C 1gcatgaaagctgagaccatcaccaggttgatcgttg  c/caaaaaaaaagtaacaggtagcagactttcaactaac 235ttgctgctattataagatgccaaaatcggcaaatcg ctggcatgaaagcttggattgtcactggtttgattgtcattcactcaaggattggacacaaga gttgctgctgctgttatgaaagctgtataChr1CMM3931650_A-T 1 acgttgcttaggtagcaccttgatttaatcaaatgc t/attgtacaggagtgtactacatccacatacaatgaa 236tagctagttgatgccaggtggcacactgcggacgga cagtagtagtagcagcagctatatactccagttgctttgtttgtcagtttccctgcattacac ctagtcgtacacaaagtataattaatcaca

TABLE 8 TRAIT RESISTANCE/TOLERANCE ATCC LINE SOURCE DESIGNATION TOINHIBITORS DEPOSIT P1003 HPPD (NON-INDUCED) R0146 HPPD SENSITIVEML0831266- HPPD PTA-13620 03093 (INDUCED + NON- Mar. 19, 2013 INDUCED)ML0831265- 09PM72399 ACCASE PTA-12933 01493 G2096S MUTATION May 31, 2012ML0831265- ACCASE PTA-13619 02283 (UNKNOWN MUTATION) Mar. 19, 2013PL1214418M2- ACCASE MUTATION PTA-121398 73009 HPPD (INDUCED + NON- Jul.18, 2014 INDUCED) PL1214418M2- ACCASE MUTATION PTA-121362 80048 HPPD(INDUCED + NON- Jun. 30, 2014 INDUCED) PL1214418M2- ACCASE-G2096S 73001HPPD INDUCED PL1214418M2- ACCASE-G2096S 73013 HPPD (NON-INDUCED)

TABLE 9 Agronomic characteristics of two lines carrying both HPPD andACCase resistance/tolerance. Days to 50% Plant Sheath DesignationHeading Height Plant Type Pubesence Color Awns yield/plant PL1214418M2-range 56-94 range 57-80 cm erect to variation of variation None 13.39 gm80048 intermediate glaborous between and smooth green and purplePL1214418M2- 85 range 84-108 cm erect to variation of variation None NA73009 intermediate glaborous between and smooth green and purple

PUBLICATIONS

All publications cited in this application are herein incorporated byreference

-   Akira, Abe et al., Genome sequencing reveals agronomically important    loci in rice using MutMap Nature Biotechnology 30, 174-178 (2012),    Published online 22 Jan. 2012.-   Wright, Mark H. et al., Genome-wide association mapping reveals a    rich genetic architecture of complex traits in Oryza sativa. Nat.    Comm 2:467|DOI: 10.1038/ncomms1467, Published Online 13 Sep. 2011.    The marker information can be accessed from The Rice Diversity Home    Page and downloading the file “44K GWAS Data”    (http://www.ricediversity.org/index.cfm).-   Zhao, Keyan et al. (2011). Genome-wide association mapping reveals a    rich genetic architecture of complex traits in Oryza sativa. Nat    Comm 2:467|DOI: 10.1038/ncomms1467, Published Online 13 Sep. 2011

The invention claimed is:
 1. A mutant rice plant resistant to both HPPDand ACCase inhibiting herbicides, wherein said mutant rice plant isproduced from seeds deposited in the ATCC under Accession numbersPTA-121398 or PTA-121362.
 2. The mutant rice plant of claim 1, whereinthe HPPD inhibiting herbicides are selected from the group consisting ofmesotrione, benzobicyclon, and combinations thereof.
 3. The mutant riceplant of claim 1, wherein the HPPD inhibiting herbicides are selectedfrom the group consisting of topramezone, tembotrione, isoxaflutole, andcombinations thereof.
 4. An F1 progeny rice plant of the mutant riceplant of claim 1, or a part thereof, wherein the F1 progeny rice plantis resistant to both HPPD and ACCase inhibiting herbicides.
 5. A tissueculture produced from protoplasts or cells from the mutant rice plant ofclaim 1, wherein said cells or protoplasts of the tissue culture areproduced from a plant part selected from the group consisting of leaves,pollen, embryos, cotyledon, hypocotyl, meristematic cells, roots, roottips, pistils, anthers, flowers, stems, glumes, and panicles.
 6. A riceplant regenerated from the tissue culture of claim 5, wherein the planthas all the morphological and physiological characteristics of themutant rice plant of claim
 1. 7. A method for controlling weeds in arice field, the method comprising: (a) having the mutant rice plant ofclaim 1, or the F1 progeny rice plant of claim 5 in the field, whereinthe mutant rice plant or the F1 progeny rice plant is resistant to aplurality of herbicides; and (b) contacting the rice field with at leastone of the plurality of the herbicides to which the mutant rice plant isresistant at levels known to kill weeds wherein the plurality ofherbicides comprise herbicides selected from the group consisting ofACCase and HPPD inhibitors.
 8. The method of claim 7, wherein the mutantrice plant or the F1 progeny rice plant is resistant to both ACCase andHPPD inhibitors.
 9. The method of claim 7, wherein the ACCase inhibitingherbicides are selected from the group consisting of aryloxyphenoxypropionate, fluazifop and quizalofop.
 10. A method to develop a riceplant resistant to a plurality of herbicides comprising HPPD and ACCaseinhibitors, the method comprising: (a) introgressing genetic materialderived from ATCC deposited lines PTA-13620 and PTA-12933 into ricelines through breeding, tissue culture or transformation, wherein saidgenetic material causes resistance to HPPD and ACCase inhibitors; (b)selecting progeny of the introgressed lines that demonstrate theplurality of resistance,: and (c) producing inbred or hybrid plants fromthe progeny.
 11. The method of claim 10, further comprising applicationof mesotrione at a rate of at least 420-630 gm ai/ha and quizalofop at arate of at least 154 gm ai/ha and up to 210 gm ai/ha, to the rice plantresistant to the plurality of herbicides comprising HPPD and ACCaseinhibitors.
 12. A method of isolating a DNA molecule with a mutationassociated with tolerance to mesotrione or other HPPD inhibitingherbicides, said method comprising isolating a DNA sequence from seedsdeposited in the ATCC under Accession number PTA-13620, wherein themutation is linked to, or located between, SNP markers with nucleic acidsequences selected from the group consisting of SEQ ID NO: 4 and SEQ IDNO: 5, the mutation causing tolerance to mesotrione or other HPPDinhibiting herbicides.
 13. A recombinant nucleic acid comprising themutant DNA molecule of claim
 12. 14. A mutant rice plant resistant toboth HPPD and ACCase inhibiting herbicides, said mutant rice comprisingthe mutant DNA molecule of claim 12 and an ACCase tolerance mutationG2096S.